BISPECIFIC NANOBODIES
20220242958 · 2022-08-04
Assignee
Inventors
- Annelies Roobrouck (Oudenaarde, BE)
- Catelijne STORTELERS (Gent, BE)
- Peter Vanlandschoot (Bellem, BE)
- Stephanie Staelens (Wevelgem, BE)
- Miguel Conde (Gent, BE)
- Hugo Soares (Loures, PT)
- Dominique Schols (Herent, BE)
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/32
CHEMISTRY; METALLURGY
C07K16/2866
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to bispecific polypeptides comprising a first and a second immunoglobulin single variable domain (ISV), wherein said first ISV binds to a first target on the surface of a cancer cell with a low affinity and, when bound inhibits a function of said first target, and a said second ISV binds to a second target on the surface of said cell with a high affinity and wherein said first target is different from said second target. The present invention further discloses methods for identifying and making the same.
Claims
1.-23. (canceled)
24. A method of decreasing the toxicity of a therapeutic immunoglobulin single variable domain (ISV) to non-target cells, the method comprising generating a fusion polypeptide comprising the therapeutic ISV and an anchoring ISV, wherein: the therapeutic ISV, when monovalent, binds to a first target on a target cell with an average dissociation constant (K.sub.D) value of between 1 nM and 200 nM; the anchoring ISV, when monovalent, binds to a second target on the target cell with an average K.sub.D value of between 0.1 pM and 10 nM; and wherein the first target and the second target are located on different antigens embedded in the membrane of the target cell, and wherein the anchoring ISV does not alter a function of the second target when bound to the second target.
25. The method according to claim 24, wherein the therapeutic ISV, when monovalent, binds to the first target with an average K.sub.D value of between 10 nM and 200 nM.
26. The method according to claim 24, wherein the therapeutic ISV, when monovalent, binds to the first target with an average K.sub.D value of about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nM.
27. The method according to claim 24, wherein the therapeutic ISV inhibits a function of the first target when bound to the first target.
28. The method according to claim 24, wherein the anchoring ISV, when monovalent, binds to the second target with an average K.sub.D value of 1 nM or less.
29. The method according to claim 24, wherein the fusion polypeptide comprises a N-terminal therapeutic ISV and a C-terminal anchoring ISV.
30. The method according to claim 24, wherein the fusion polypeptide comprises a N-terminal anchoring ISV and a C-terminal therapeutic ISV.
31. The method according to claim 25, wherein the fusion polypeptide further comprises a linker that separates the therapeutic ISV and the anchoring ISV.
32. The method according to claim 24, wherein the first target is a protein antigen.
33. The method according to claim 24, wherein the protein antigen is a cellular receptor.
34. The method according to claim 24, wherein the second target is a protein antigen.
35. The method according to claim 24, wherein the target cell is a diseased cell, and wherein the first target and the second target are disease-associated antigens.
36. The method according to claim 24, wherein the target cell is a cancer cell, and wherein the first target and the second target are tumor-associated antigens.
37. The method according to claim 24, wherein the fusion polypeptide further comprises a drug, optionally wherein the drug is a toxin or toxin moiety.
38. The method according to claim 24, wherein the fusion polypeptide further comprises an imaging agent.
39. The method according to claim 38, wherein the imaging agent is selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a metal complex, a metal, colloidal gold, a metallic label, chemiluminescent, bioluminescent, a chromophore, and a mixture thereof.
40. The method according to claim 24, wherein: the first target is chosen from the group consisting of a Receptor Tyrosine Kinase, a G-Protein-Coupled Receptor (GPCR), DDR1, Discoidin I (CD167a antigen), DDR2, ErbB-1, c-ErbB-2, FGFR-1, FGFR-3, CD135 antigen, CD117 antigen, Protein tyrosine kinase-1, c-Met, CD148 antigen, c-Ret, ROR1, ROR2, Tie-1, Tie-2, CD202b antigen, Trk-A, Trk-B, Trk-C, VEGFR-1, VEGFR-2, VEGFR-3, Notch receptor 1-4, FAS receptor, DR5, DR4, CD47, CD4, CX3CR1, CXCR3, CXCR4, CXCR7, Chemokine binding protein 2, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2), and Interleukin-23 receptor (IL23R), optionally wherein the Receptor Tyrosine Kinase is a class I Receptor Tyrosine Kinase; and the second target is chosen from the group consisting of carcinoembryonic antigen (CEA), MART-1, gp100, MAGE-1, HER-2, LewisY antigen, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, EpCAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD147, a growth factor receptors, and a Cytokine receptor, optionally wherein the growth factor receptor is ErbB3 or ErbB4, and optionally wherein the Cytokine receptor is Interleukin-2 receptor gamma chain (CD132 antigen), Interleukin-10 receptor alpha chain (IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B), IL-12R-beta1, IL-12 receptor beta-2, Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213 al antigen), Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding protein), Interleukin-17 receptor (IL-17 receptor), Interleukin-17B receptor (IL-17B receptor), Interleukin 21 receptor precursor (IL-21R), Interleukin-1 receptor, type I (IL-1R-1) (CD121a), Interleukin-1 receptor, type II (IL-1R-beta) (CDw121b), Interleukin-1 receptor antagonist protein (IL-1ra), Interleukin-2 receptor alpha chain (CD25 antigen), Interleukin-2 receptor beta chain (CD122 antigen), or Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen).
41. The method according to claim 24, wherein the first target and the second target are chosen from the group consisting of: EGFR as the first target and carcinoembryonic antigen (CEA) as the second target; Receptor Tyrosine Kinase as the first target and a tumor-associated antigen (TAA) as the second target; a G-Protein-Coupled Receptor (GPCR) as the first target and a hematopoietic differentiation antigen as the second target; Receptor Tyrosine Kinase as the first target and a hematopoietic differentiation antigen as the second target; a GPCR as the first target and a TAA as the second target; CXCR4 as the first target and CD123 as the second target; DR5 as the first target and EpCam as the second target; DR4 as the first target and EpCam as the second target; CD95 as the first target and EpCam as the second target; CD47 as the first target and CD123 as the second target; CD47 as the first target and EpCam as the second target; CD4 as the first target and CXCR4 as the second target; Interleukin-12 receptor beta-1 chain (IL-12R-beta1) as the first target and CD4 as the second target; Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2) as the first target and CD4 as the second target; and Interleukin-23 receptor (IL23R) as the first target and CD4 as the second target.
42. The method according to claim 24, wherein the K.sub.D is measured by surface plasmon resonance.
43. A method of decreasing the toxicity of a therapeutic immunoglobulin single variable domain (ISV) to non-target cells, the method comprising generating a fusion polypeptide comprising a therapeutic ISV and an anchoring ISV, wherein: the therapeutic ISV, when monovalent, binds to a first target with an average EC50 value of between 10 nM and 200 nM; the anchoring ISV, when monovalent, binds to a second target with an average EC50 value of between 10 nM and 0.1 pM; and wherein the first target and the second target are located on different antigens embedded in the membrane of the target cell, and wherein the anchoring ISV does not alter a function of the second target when bound to the second target.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DESCRIPTION OF THE INVENTION
[0087] Immunoglobulin sequences, such as antibodies and antigen binding fragments derived there from (e.g., immunoglobulin single variable domains or ISVs) are used to specifically target their respective antigens in research and therapeutic applications. The generation of immunoglobulin single variable domains such as e.g., VHHs or Nanobodies may involve the immunization of an experimental animal such as a Llama, construction of phage libraries from immune tissue, selection of phage displaying antigen binding immunoglobulin single variable domains and screening of said domains and engineered constructs thereof for the desired specificities (WO 94/04678). Alternatively, similar immunoglobulin single variable domains such as e.g., dAbs can be generated by selecting phage displaying antigen binding immunoglobulin single variable domains directly from naive or synthetic libraries and subsequent screening of said domains and engineered constructs thereof for the desired specificities (Ward et al., Nature, 1989, 341: 544-6; Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd.). Unfortunately, the use of monoclonal and/or heavily engineered antibodies also carries a high manufacturing cost and may result in suboptimal tumor penetration compared to other strategies.
[0088] Definitions: [0089] a) Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks mentioned in paragraph a) on page 46 of WO 08/020079. [0090] b) Unless indicated otherwise, the term “immunoglobulin single variable domain” or “ISV” is used as a general term to include but not limited to antigen-binding domains or fragments such as V.sub.HH domains or V.sub.H or V.sub.L domains, respectively. The terms antigen-binding molecules or antigen-binding protein are used interchangeably and include also the term Nanobodies. The immunoglobulin single variable domains can be light chain variable domain sequences (e.g., a V.sub.L-sequence), or heavy chain variable domain sequences (e.g., a V.sub.H-sequence); more specifically, they can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. Accordingly, the immunoglobulin single variable domains can be domain antibodies, or immunoglobulin sequences that are suitable for use as domain antibodies, single domain antibodies, or immunoglobulin sequences that are suitable for use as single domain antibodies, “dAbs”, or immunoglobulin sequences that are suitable for use as dAbs, or Nanobodies, including but not limited to V.sub.HH sequences. The invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The immunoglobulin single variable domain includes fully human, humanized, otherwise sequence optimized or chimeric immunoglobulin sequences. The immunoglobulin single variable domain and structure of an immunoglobulin single variable domain can be considered—without however being limited thereto—to be comprised of four framework regions or “FR's”, which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”, respectively; which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively. It is noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx N.V. and thus may also be referred to as Nanobody® or Nanobodies®, respectively. [0091] c) Unless indicated otherwise, the terms “immunoglobulin sequence”, “sequence”, “nucleotide sequence” and “nucleic acid” are as described in paragraph b) on page 46 of WO 08/020079. [0092] d) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins. [0093] e) Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is made to Table A-2 on page 48 of the International application WO 08/020079 of Ablynx N.V. entitled “Immunoglobulin single variable domains directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with IL-6 mediated signalling”. [0094] f) For the purposes of comparing two or more nucleotide sequences, the percentage of “sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated or determined as described in paragraph e) on page 49 of WO 08/020079 (incorporated herein by reference), such as by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence—compared to the first nucleotide sequence—is considered as a difference at a single nucleotide (position); or using a suitable computer algorithm or technique, again as described in paragraph e) on pages 49 of WO 08/020079 (incorporated herein by reference). [0095] g) For the purposes of comparing two or more immunoglobulin single variable domains or other amino acid sequences such e.g. the polypeptides of the invention etc., the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence (also referred to herein as “amino acid identity”) may be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08/020079 (incorporated herein by reference), such as by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence—compared to the first amino acid sequence—is considered as a difference at a single amino acid residue (position), i.e., as an “amino acid difference” as defined herein; or using a suitable computer algorithm or technique, again as described in paragraph f) on pages 49 and 50 of WO 08/020079 (incorporated herein by reference).
[0096] Also, in determining the degree of sequence identity between two immunoglobulin single variable domains, the skilled person may take into account so-called “conservative” amino acid substitutions, as described on page 50 of WO 08/020079.
[0097] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V.sub.HH domain from a llama is for example given by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional V.sub.H domains form the V.sub.H/V.sub.L interface and potential camelizing substitutions on these positions can be found in the prior art cited above. [0098] h) Immunoglobulin single variable domains and nucleic acid sequences are said to be “exactly the same” if they have 100% sequence identity (as defined herein) over their entire length. [0099] i) When comparing two immunoglobulin single variable domains, the term “amino acid difference” refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two immunoglobulin single variable domains can contain one, two or more such amino acid differences. [0100] j) When a nucleotide sequence or amino acid sequence is said to “comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of” another nucleotide sequence or amino acid sequence, this has the meaning given in paragraph i) on pages 51-52 of WO 08/020079. [0101] k) The term “in essentially isolated form” has the meaning given to it in paragraph j) on pages 52 and 53 of WO 08/020079. [0102] l) The terms “domain” and “binding domain” have the meanings given to it in paragraph k) on page 53 of WO 08/020079. [0103] m) The terms “antigenic determinant” and “epitope”, which may also be used interchangeably herein, have the meanings given to it in paragraph l) on page 53 of WO 08/020079. [0104] n) As further described in paragraph m) on page 53 of WO 08/020079, an amino acid sequence (such as an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be “against” or “directed against” said antigenic determinant, epitope, antigen or protein. [0105] o) The term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an ISV, Nanobody or a polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity.
[0106] The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K.sub.D or KD), is a measure for the binding strength between an antigenic determinant, i.e. the target, and an antigen-binding site on the antigen-binding protein, i.e. the ISV or Nanobody: the lesser the value of the K.sub.D, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K.sub.A), which is 1/K.sub.D). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest.
[0107] Avidity is the affinity of the polypeptide, i.e. the ligand is able to bind via two (or more) pharmacophores (ISV) in which the multiple interactions synergize to enhance the “apparent” affinity. Avidity is the measure of the strength of binding between the polypeptide of the invention and the pertinent antigens. The polypeptide of the invention is able to bind via its two (or more) building blocks, such as ISVs or Nanobodies, to the at least two targets, in which the multiple interactions, e.g. the first building block, ISV or Nanobody binding to the first target and the second building block, ISV, or Nanobody binding to the second target, synergize to enhance the “apparent” affinity. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecules. For example, and without limitation, polypeptides that contain two or more building blocks, such as ISVs or Nanobodies directed against different targets on a cell and in particular against human CXCR4 and human CD123 may (and usually will) bind with higher avidity than each of the individual monomers or individual building blocks, such as, for instance, the monovalent ISVs or Nanobodies, comprised in the polypeptides of the invention.
[0108] In the present invention, monovalent antigen-binding proteins (such as the building blocks, ISVs, amino acid sequences, Nanobodies and/or polypeptides of the invention) are said to bind to their antigen with a high affinity when the dissociation constant (K.sub.D) is 10.sup.−9 to 10.sup.−12 moles/liter or less, and preferably 10.sup.−10 to 10.sup.−12 moles/liter or less and more preferably 10.sup.−11 to 10.sup.−12 moles/liter (i.e. with an association constant (K.sub.A) of 10.sup.9 to 10.sup.12 liter/moles or more, and preferably 10.sup.10 to 10.sup.12 liter/moles or more and more preferably 10.sup.11 to 10.sup.12 liter/moles).
[0109] In the present invention, monovalent antigen-binding proteins (such as the building blocks, ISVs, amino acid sequences, Nanobodies and/or polypeptides of the invention) are said to bind to their antigen with a low affinity when the dissociation constant (K.sub.D) is 10.sup.−6 to 10.sup.−9 moles/liter or more, and preferably 10.sup.−6 to 10.sup.−8 moles/liter or more and more preferably 10.sup.−6 to 10.sup.−7 moles/liter (i.e. with an association constant (K.sub.A) of 10.sup.6 to 10.sup.9 liter/moles or more, and preferably 10.sup.6 to 10.sup.8 liter/moles or more and more preferably 10.sup.6 to 10.sup.7 liter/moles).
[0110] A medium affinity can be defined as values ranging in between high-low, e.g. 10.sup.−8 to 10.sup.−10 moles/liter.
[0111] Any K.sub.D value greater than 10.sup.−4 mol/liter (or any K.sub.A value lower than 10.sup.4 M.sup.−1) liters/mol is generally considered to indicate non-specific binding.
[0112] The polypeptides of the invention comprise a first and a second building block, e.g. a first and a second ISV, or a first and a second Nanobody. Preferably the affinity of each building block, e.g. ISV or Nanobody, is determined individually. In other words, the affinity is determined for the monovalent building block, ISV or Nanobody, independent of avidity effects due to the other building block, ISV or Nanobody, which might or might not be present. The affinity for a monovalent building block, ISV or Nanobody can be determined on the monovalent building block, ISV or Nanobody per se, i.e. when said monovalent building block, ISV or Nanobody is not comprised in the polypeptide of the invention. In the alternative or in addition, the affinity for a monovalent building block, ISV or Nanobody can be determined on one target while the other target is absent.
[0113] The binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
[0114] The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more than 10.sup.−4 moles/liter or 10.sup.−3 moles/liter (e.g., of 10.sup.−2 moles/liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (K.sub.A), by means of the relationship [K.sub.D=1/K.sub.A].
[0115] The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the K.sub.D, or dissociation constant, which has units of mol/liter (or M). The affinity can also be expressed as an association constant, K.sub.A, which equals 1/K.sub.D and has units of (mol/liter).sup.−1 (or M.sup.−1). In the present specification, the stability of the interaction between two molecules (such as an amino acid sequence, Nanobody or polypeptide of the invention and its intended target) will mainly be expressed in terms of the K.sub.D value of their interaction; it being clear to the skilled person that in view of the relation K.sub.A=1/K.sub.D, specifying the strength of molecular interaction by its K.sub.D value can also be used to calculate the corresponding K.sub.A value. The K.sub.D-value characterizes the strength of a molecular interaction also in a thermodynamic sense as it is related to the free energy (DG) of binding by the well known relation DG=RT.In(K.sub.D) (equivalently DG=−RT.In(K.sub.A)), where R equals the gas constant, T equals the absolute temperature and In denotes the natural logarithm.
[0116] The K.sub.D for biological interactions which are considered meaningful (e.g. specific) are typically in the range of 10.sup.−10M (0.1 nM) to 10.sup.−5M (10000 nM). The stronger an interaction is, the lower is its K.sub.D.
[0117] The K.sub.D can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as k.sub.off, to the rate of its association, denoted k.sub.on (so that K.sub.D=k.sub.off/k.sub.on and K.sub.A=k.sub.on/k.sub.off). The off-rate k.sub.off has units s.sup.−1 (where s is the SI unit notation of second). The on-rate k.sub.on has units M.sup.−1s.sup.−1. The on-rate may vary between 10.sup.2 M.sup.−1s.sup.−1 to about 10.sup.7 M.sup.−1s.sup.−1, approaching the diffusion-limited association rate constant for bimolecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation t.sub.1/2=In(2)/k.sub.off. The off-rate may vary between 10.sup.−6s.sup.−1 (near irreversible complex with a t.sub.1/2 of multiple days) to 1 s.sup.−1 (t.sub.1/2=0.69 s).
[0118] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al., Intern. Immunology, 13, 1551-1559, 2001). The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k.sub.on, k.sub.off measurements and hence K.sub.D (or K.sub.A) values. This can for example be performed using the well-known BIAcoreφ system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.
[0119] It will also be clear to the skilled person that the measured K.sub.D may correspond to the apparent K.sub.D if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent K.sub.D may be measured if one molecule contains more than one recognition site for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.
[0120] Another approach that may be used to assess affinity is the 2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes a solution phase binding equilibrium measurement and avoids possible artefacts relating to adsorption of one of the molecules on a support such as plastic.
[0121] However, the accurate measurement of K.sub.D may be quite labour-intensive and as consequence, often apparent K.sub.D values are determined to assess the binding strength of two molecules. It should be noted that as long all measurements are made in a consistent way (e.g. keeping the assay conditions unchanged) apparent K.sub.D measurements can be used as an approximation of the true K.sub.D and hence in the present document K.sub.D and apparent K.sub.D should be treated with equal importance or relevance.
[0122] Finally, it should be noted that in many situations the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule. For example, to assess the binding strength between molecules A and B, one may e.g. use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B. As a result an IC.sub.50 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved. Provided K.sub.D ref, the K.sub.D of the reference molecule, is known, as well as the total concentration c.sub.ref of the reference molecule, the apparent K.sub.D for the interaction A-B can be obtained from following formula: K.sub.D=IC.sub.50/(1+c.sub.ref/K.sub.D ref). Note that if c.sub.ref<<K.sub.D ref, K.sub.D≈IC.sub.50. Provided the measurement of the IC.sub.50 is performed in a consistent way (e.g. keeping c.sub.ref fixed) for the binders that are compared, the strength or stability of a molecular interaction can be assessed by the IC.sub.50 and this measurement is judged as equivalent to K.sub.D or to apparent K.sub.D throughout this text. [0123] p) The half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as described in paragraph o) on page 57 of WO 08/020079 and as mentioned therein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally be as described in paragraph o) on page 57 of WO 08/020079. As also mentioned in paragraph o) on page 57 of WO 08/020079, the half-life can be expressed using parameters such as the t½-alpha, t½-beta and the area under the curve (AUC). Reference is for example made to the Experimental Part below, as well as to the standard handbooks, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The terms “increase in half-life” or “increased half-life” as also as defined in paragraph o) on page 57 of WO 08/020079 and in particular refer to an increase in the t½-beta, either with or without an increase in the t½-alpha and/or the AUC or both. [0124] q) In respect of a target or antigen, the term “interaction site” on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an “interaction site” can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein). [0125] r) An immunoglobulin single variable domain or polypeptide is said to be “specific for” a first target or antigen compared to a second target or antigen when it binds to the first antigen with an affinity/avidity (as described above, and suitably expressed as a K.sub.D value, K.sub.A value, K.sub.off rate and/or K.sub.on rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10.000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide. For example, the first antigen may bind to the target or antigen with a K.sub.D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10,000 times less or even less than that, than the K.sub.D with which said amino acid sequence or polypeptide binds to the second target or polypeptide. Preferably, when an immunoglobulin single variable domain or polypeptide is “specific for” a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen. [0126] s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are used interchangeably herein to mean the ability of an immunoglobulin single variable domain or polypeptide to interfere with the binding of the natural ligand to its receptor(s). The extent to which an immunoglobulin single variable domain or polypeptide of the invention is able to interfere with the binding of another compound such as the natural ligand to its target, e.g., CXCR4, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative cross-blocking assay uses a FACS- or an ELISA-based approach or Alphascreen to measure competition between the labelled (e.g., His tagged or biotinylated) immunoglobulin single variable domain or polypeptide according to the invention and the other binding agent in terms of their binding to the target. The experimental part generally describes suitable FACS-, ELISA- or Alphascreen-displacement-based assays for determining whether a binding molecule cross-blocks or is capable of cross-blocking an immunoglobulin single variable domain or polypeptide according to the invention. It will be appreciated that the assay can be used with any of the immunoglobulin single variable domains or other binding agents described herein. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the invention is for example one which will bind to the target in the above cross-blocking assay such that, during the assay and in the presence of a second amino acid sequence or other binding agent of the invention, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the invention is between 60% and 100% (e.g., in ELISA/Alphascreen based competition assay) or between 80% to 100% (e.g., in FACS based competition assay) of the maximum theoretical displacement (e.g. displacement by cold (e.g., unlabeled) immunoglobulin single variable domain or polypeptide that needs to be cross-blocked) by the to be tested potentially cross-blocking agent that is present in an amount of 0.01 mM or less (cross-blocking agent may be another conventional monoclonal antibody such as IgG, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (e.g., diabodies, triabodies, minibodies, VHHs, dAbs, VHs, VLs). [0127] t) An amino acid sequence such as e.g. an immunoglobulin single variable domain or polypeptide according to the invention is said to be a “VHH1 type immunoglobulin single variable domain” or “VHH type 1 sequence”, if said VHH1 type immunoglobulin single variable domain or VHH type 1 sequence has 85% identity (using the VHH1 consensus sequence as the query sequence and use the blast algorithm with standard setting, i.e., blosom62 scoring matrix) to the VHH1 consensus sequence (QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA), and mandatorily has a cysteine in position 50, i.e., C50 (using Kabat numbering). [0128] u) An amino acid sequence such as e.g., an immunoglobulin single variable domain or polypeptide according to the invention is said to be “cross-reactive” for two different antigens or antigenic determinants (such as serum albumin from two different species of mammal, such as human serum albumin and cynomolgus monkey serum albumin) if it is specific for (as defined herein) both these different antigens or antigenic determinants. [0129] v) As further described in paragraph q) on pages 58 and 59 of WO 08/020079 (incorporated herein by reference), the amino acid residues of an immunoglobulin single variable domain are numbered according to the general numbering for V.sub.H domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to V.sub.HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195 (see for example
[0132] The term half maximal effective concentration (EC50) refers to the concentration of a compound which induces a response halfway between the baseline and maximum after a specified exposure time. In the present context it is used as a measure of a polypeptide's, ISV's or Nanobody's potency. The EC50 of a graded dose response curve represents the concentration of a compound where 50% of its maximal effect is observed. Concentration is preferably expressed in molar units.
[0133] In biological systems, small changes in ligand concentration typically result in rapid changes in response, following a sigmoidal function. The inflection point at which the increase in response with increasing ligand concentration begins to slow is the EC50. This can be determined mathematically by derivation of the best-fit line. Relying on a graph for estimation is convenient in most cases. In case the EC50 is provided in the examples section, the experiments were designed to reflect the KD as accurate as possible. In other words, the EC50 values may then be considered as KD values. The term “average KD” relates to the average KD value obtained in at least 1, but preferably more than 1, such as at least 2 experiments. The term “average” refers to the mathematical term “average” (sums of data divided by the number of items in the data).
[0134] It is also related to IC50 which is a measure of a compound's inhibition (50% inhibition). For competition binding assays and functional antagonist assays IC50 is the most common summary measure of the dose-response curve. For agonist/stimulator assays the most common summary measure is the EC50.
[0135] Bispecific Polypeptides
[0136] The present invention relates to particular polypeptides, also referred to as “polypeptides of the invention” that comprise or essentially consist of (i) a first building block consisting essentially of a first immunoglobulin single variable domain, wherein said first immunoglobulin single variable domain binds a first target on the surface of a cell with low affinity, but when bound impairs or inhibits a function of said first target (functional ISV); and (ii) a second building block consisting essentially of a second immunoglobulin single variable domain, wherein said second immunoglobulin single variable domain binds a second target on the surface of a cell with high affinity, but when bound impairs or inhibits the function of said second target preferably only minimally (anchoring ISV). In addition or alternatively, the function of said second target is preferably not vital to the cell, e.g. redundant. Consequently, inhibiting the function of said second target (the “anchor”) will result in limited or negligible side-effects and/or toxicity. Nevertheless, inhibiting the function of only said second target (anchor) on normal cells, i.e. without inhibiting the function of said first target, is already a significant reduction of the toxicity and side-effects when compared to a treatment using high affinity antibodies against either one or both targets. The polypeptides of the present invention provide a more specific inhibition of tumor proliferation and arrest or killing of the tumor cells than prior art antibodies. Preferably, the bispecific polypeptides of the invention comprise at least two binding moieties, such as for instance two building blocks, ISVs or Nanobodies, wherein at least the first binding moiety (functional ISV) is specific for a tumor associated antigen (e.g., an antigen expressed on a tumor cell, also called ‘tumor marker’). The terms bispecific polypeptide, bispecific and bispecific antibody are used interchangeably herein.
[0137] Accordingly, the present invention relates to a polypeptide comprising a first (functional) and a second (anchoring) immunoglobulin single variable domain (ISV), [0138] wherein said first ISV (functional ISV), binds to a first target with a low affinity; [0139] said second ISV (anchoring ISV) binds to a second target with a high affinity; and
[0140] wherein said first target and said second target are present on the surface of a cell and wherein said first target is different from said second target, and optionally said first building block (functional building block or anchoring ISV) and said second building block (anchoring building block or anchoring ISV) are linked via a linker.
[0141] The polypeptides of the invention are designed to reduce or impair a contribution of the first target to the disorder, e.g. a malignant process. The terms “malignant process” and “malignancy” are used interchangeably herein. In the present context, malignancy is the tendency of a medical condition, especially tumors, to become progressively worse and to potentially result in death. Malignancy is characterized by anaplasia, invasiveness, and/or metastasis. The pharmacologic effect of the polypeptides of the invention therefore will reside eventually in inhibiting or impairing at least one, but preferably more than one of anaplasia, invasiveness, metastasis, proliferation, differentiation, migration and/or survival of said cell. The pharmacologic effect of the polypeptides of the invention therefore will reside in increasing or supporting at least one, but preferably more than one of apoptosis, cell killing and/or growth arrest of said cell. The phenomena characterized by these terms are well known in the art.
[0142] The bispecific or multispecific polypeptides of the present invention comprise or essentially consist of at least two building blocks, e.g. ISVs, of which the first building block, e.g. the first ISV, has an increased affinity for its antigen, i.e. the first target, upon binding by the second building block, e.g. the second ISV, to its antigen, i.e. the second target. Such increased affinity (apparent affinity), due to avidity effects, is also called ‘conditional bispecific or multispecific binding’. Such bispecific or multispecific polypeptide is also called ‘a conditionally binding bispecific or multispecific polypeptide of the invention’.
[0143] It will be appreciated that the order of the first building block and the second building block on the polypeptide (orientation) can be chosen according to the needs of the person skilled in the art, as well as the relative affinities which may depend on the location of these building blocks in the polypeptide, and whether the polypeptide comprises a linker, is a matter of design choice. However, some orientations, with or without linkers, may provide preferred binding characteristics in comparison to other orientations. For instance, the order of the first and the second building block in the polypeptide of the invention can be (from N-terminus to C-terminus): (i) first building block (e.g. a first ISV such as a first Nanobody)—[linker]—second building block (e.g. a second ISV such as a second Nanobody); or (ii) second building block (e.g. a second ISV such as a second Nanobody)—[linker]—first building block (e.g. a first ISV such as a first Nanobody); (wherein the linker is optional). All orientations are encompassed by the invention, and polypeptides that contain an orientation that provides desired binding characteristics can be easily identified by routine screening, for instance as exemplified in the examples section.
[0144] Binding of the second antigen by the second, anchoring ISV enhances binding of the first antigen by the first, functional ISV of said at least two ISVs, as a result the potency of the first, functional ISV, such as Nanobody comprised in the bispecific polypeptide is increased compared to the corresponding monovalent ISV, e.g. a Nanobody.
[0145] As used herein, the term “potency” is a measure of an agent, such as a polypeptide, ISV or Nanobody, its biological activity. Potency of an agent can be determined by any suitable method known in the art, such as for instance as described in the examples section. Cell culture based potency assays are often the preferred format for determining biological activity since they measure the physiological response elicited by the agent and can generate results within a relatively short period of time. Various types of cell based assays, based on the mechanism of action of the product, can be used, including but not limited to proliferation assays, cytotoxicity assays, reporter gene assays, cell surface receptor binding assays and assays to measure induction/inhibition of functionally essential protein or other signal molecule (such as phosphorylated proteins, enzymes, cytokines, cAMP and the like), all well known in the art. Results from cell based potency assays can be expressed as “relative potency” as determined by comparison of the bispecific polypeptide of the invention to the response obtained for the corresponding reference monovalent ISV, e.g. a polypeptide comprising only one ISV or one Nanobody, optionally further comprising an irrelevant Nanobody, such as Cablys (cf. examples section).
[0146] A compound, e.g. the bispecific polypeptide, is said to be more potent than the reference compound, e.g. the corresponding monovalent or monospecific ISV or Nanobody or polypeptide comprising the corresponding monovalent or monospeciic ISV or Nanobody, when the response obtained for the compound, e.g. the bispecific polypeptide, is at least 2 times, but preferably at least 3 times, such as at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times, and even more preferably even at least 200 times, or even at least 500 times, or even 1000 times better (e.g. functionally better) than the response by the reference compound, e.g. the corresponding monovalent ISV or Nanobody in a given assay.
[0147] The cell of the invention relates in particular to mammalian cells, and preferably to primate cells and even more preferably to human cells. The cell is preferably a cancer cell, wherein said cancer is as defined herein, preferably a leukaemia, and even more preferably AML.
[0148] The membrane (also called plasma membrane or phospholipid bilayer) surrounds the cytoplasm of a cell, which is the outer boundary of the cell, i.e. the membrane is the surface of the cell. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of phospholipids. Embedded within this membrane is a variety of protein molecules, such as channels, pumps and cellular receptors. Since the membrane is fluid, the protein molecules can travel within the membrane.
[0149] First Building Block (Functional Building Block)
[0150] As described herein, a polypeptide of the invention contains at least two building blocks, such as ISVs or Nanobodies of the invention of which the first building block, ISV or Nanobody is directed against a first target involved in a disease or disorder, such as a malignancy, and in particular involved in a leukaemia such as AML, and even more particularly against human CXCR4. Preferably, said first target is unique to a diseased cell, e.g. a cancer cell, e.g. said first target is not expressed on a normal cell. However, this will not be the case generally. In most cases, said first target will be present on both normal and diseased cells, such as cancer cells. Hence, to increase specificity to the diseased cell, e.g. cancer cell and/or decrease side-effects and toxicity due to e.g. binding to normal cells, the first building block, ISV or Nanobody in such polypeptides will bind to said first target and in particular human CXCR4, with increased avidity compared to the corresponding monomer or monovalent building block, ISV or Nanobody of the invention when both the first and second target are present on a cell, preferably a cancer cell (cis-format). When bound to the first target, said first, functional building block, ISV or Nanobody will inhibit a function of said first target.
[0151] A function of a target relates to any change in a measurable biological or biochemical property elicited by said target, including physiological changes of the cell such as changes in proliferation, differentiation, anaplasia, invasiveness, metastasis, migration, survival, apoptosis, transport processes, metabolism, motility, cytokine release, cytokine composition, second messengers, enzymes, receptors, etc. Preferably the function of a target is determined by cell culture based potency assays as described above.
[0152] It will be appreciated that due to its low affinity, the function of said first building block, ISV or Nanobody cannot be tested or ascertained directly in all cases. The present inventors demonstrated that it is nonetheless possible to test low affinity binders which impair or inhibit the function of their cognate targets (see examples section). For instance, the present inventors used family members of a previously identified high affinity member and mutated this in order to decrease the affinity. By using family members, it was ascertained that the same epitope on the target was bound. The term “family” as used in the present specification refers to a group of ISV, Nanobody and/or VHH sequences that have identical lengths (i.e. they have the same number of amino acids within their sequence) and of which the amino acid sequence between position 8 and position 106 (according to Kabat numbering) has an amino acid sequence identity of 89% or more. Family members are derived from a common ancestor during the B cell maturation process.
[0153] When, designing the polypeptides of the invention, the first building block, e.g. the first ISV, is chosen for its low affinity per se, disregarding the influence of any avidity effects.
[0154] Accordingly the present invention relates to a polypeptide as described herein, wherein said first ISV binds to a first target with an average KD value of between 1 nM and 200 nM, such as an average KD value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nM, or 200 nM. Preferably, the KD is determined by SPR.
[0155] In a further aspect, the present invention relates to a polypeptide as described herein, wherein said first ISV has a low affinity when measured as a monovalent.
[0156] The present invention also relates to a polypeptide as described herein, wherein said first ISV binds to a first target on the surface of a cell with an EC50 value of between 1 nM and 200 nM, such as an average EC50 value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nM.
[0157] Accordingly the present invention relates to a polypeptide as described herein, wherein said average EC50 is measured on cells comprising said target 1 but substantially lacking said target 2.
[0158] The present invention relates also to a polypeptide as described herein, wherein said average KD is determined (indirectly) by any technique known in the art, such as for instance SPR, FACS, or ELISA on a monovalent first ISV.
[0159] The first ISV of the invention may for example be directed against a first antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of said first target, such as, for instance, a Receptor Tyrosine Kinase (RTK) or a G-protein coupled receptor (GPCR) participating in malignancy, and in particular human CXCR4 (OMIM 162643). If the first building block, such as an ISV or Nanobody binds to said first target a function of said first target is impaired or inhibited.
[0160] The first target of the invention can be any target, such as a cellular receptor, on the surface of a cell which is known to participate in malignancy.
[0161] For instance, receptor tyrosine kinases (RTK) and RTK-mediated signal transduction pathways are involved in tumour initiation, maintenance, angiogenesis, and vascular proliferation. About 20 different RTK classes have been identified, of which the most extensively studied are: 1. RTK class I (EGF receptor family) (ErbB family), 2. RTK class II (Insulin receptor family), 3. RTK class III (PDGF receptor family), 4. RTK class IV (FGF receptor family), 5. RTK class V (VEGF receptors family), 6. RTK class VI (HGF receptor family), 7. RTK class VII (Trk receptor family), 8. RTK class VIII (Eph receptor family), 9. RTK class IX (AXL receptor family), 10. RTK class X (LTK receptor family), 11. RTK class XI (TIE receptor family), 12. RTK class XII (ROR receptor family), 13. RTK class XIII (DDR receptor family), 14. RTK class XIV (RET receptor family), 15. RTK class XV (KLG receptor family), 16. RTK class XVI (RYK receptor family), 17. RTK class XVII (MuSK receptor family). In particular, targets such as epidermal growth factor receptors (EGFR), platelet-derived growth factor receptors (PDGFR), vascular endothelial growth factor receptors (VEGFR), c-Met, HER3, plexins, integrins, CD44, RON and on receptors involved in pathways such as the Ras/Raf/mitogen-activated protein (MAP)-kinase and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways.
[0162] Furthermore, a tight operational relationship occurs between GPCRs and other receptors responding to growth factors. GPCRs signaling may precede, follow, parallel or synergize the signaling of receptors for steroids, epidermal growth factor (EGF), platelet derived growth factor (PDGF), etc. In lung, gastric, colorectal, pancreatic and prostatic cancers, sustained GPCRs stimulation is promoted by activatory autocrine and paracrine loops.
[0163] There are two principal signal transduction pathways involving the G protein-coupled receptors: the cAMP signal pathway and the phosphatidylinositol signal pathway, both of which can participate in malignancy. When a ligand binds to the GPCR it causes a conformational change in the GPCR, which allows it to act as a guanine nucleotide exchange factor (GEF). The GPCR can then activate an associated G-protein by exchanging its bound GDP for a GTP. The G-protein's α subunit, together with the bound GTP, can then dissociate from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins directly depending on the α subunit type (Gαs, Gαi/o, Gαq/11, Gα12/13). Hence, the eventual functions of said first target are signal transduction, e.g. the transmission and processing of cues from the outside environment to the inside of the cell, upon which the cell reacts. In cancer cells, the normal process is altered.
[0164] Preferably, the first target is chosen from Discoidin domain receptor (DDR), a receptor tyrosine kinase that is distinguished by a unique extracellular domain homologous to the lectin Discoidin I (CD167a antigen), DDR2, ErbB-1, C-erbB-2, FGFR-1, FGFR-3, CD135 antigen, CD 117 antigen, Protein tyrosine kinase-1, c-Met, CD148 antigen, C-ret, ROR1, ROR2, Tie-1, Tie-2, CD202b antigen, Trk-A, Trk-B, Trk-C, VEGFR-1, VEGFR-2, VEGFR-3, Notch receptor 1-4, FAS receptor, DR5, DR4, CD47, CX3CR1, CXCR-3, CXCR-4, CXCR-7, Chemokine binding protein 2, and CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11.
[0165] Accordingly, the present invention relates to polypeptides of the invention wherein the first building block, ISV or Nanobody inhibits of impairs at least one function, preferably more than one, and most preferably all functions of said first target.
[0166] Preferably, the first ISV is directed against an interaction site of said first target, thereby impairing a function of said first target. A preferred interaction site for binding by the first ISV of the invention is a ligand binding site on the first target. For instance, binding of the anti-CXCR4 ISV of the invention may inhibit or displace binding of the cognate ligand, i.e. SDF-1 (also known as CXCL12) to CXCR4. Also, when the first target is part of a binding pair (for example, a receptor-ligand binding pair), the immunoglobulin single variable domains and polypeptides may be such that they compete with the cognate binding partners, e.g., SDF-1 for binding with CXCR4 or HGF for binding to c-Met, and/or such that they (fully or partially) neutralize binding of the cognate binding partner to the target. Also, when a ligand, e.g. SDF-1 associates with other proteins or polypeptides, such as to form protein complexes (e.g., with CXCR4) it is within the scope of the invention that the immunoglobulin single variable domains and polypeptides of the invention bind to the receptor associated with its ligand, e.g. SDF-1 associated with CXCR4, provided a function of the receptor is impaired. In all these cases, the immunoglobulin single variable domains and polypeptides of the invention may bind to such associated protein complexes with an affinity and/or specificity that may be the same as or different from (i.e., higher than or lower than) the affinity and/or specificity with which the immunoglobulin single variable domains and polypeptides of the invention bind to the cellular target, e.g. receptor and in particular human CXCR4 in its non-associated state, again provided a function of the first target is inhibited.
[0167] Since various cell surface receptors require dimerization for activation, it is preferred that in such cases the first ISV of the invention binds to these dimerization sites, such as homo- or hetero-dimerization sites, thereby inhibiting or preventing dimerization and thus signalling by the receptor pair.
[0168] Furthermore, most receptors exist in various conformations, e.g. the relaxed conformation binds substrates readily, while upon binding of a substrate the conformation is changed allowing signalling. Accordingly, the first ISV of the invention may also impair the function of the first target by allosteric effects. For instance, binding of the first ISV prevents the first target from conformational changes, thereby inhibiting signalling.
[0169] Advantageously, since the bispecific constructs of the invention are directed against two different targets, inadvertent dimerization and thus signalling is precluded.
[0170] It is also expected that the immunoglobulin single variable domains and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of its targets; or at least to those analogs, variants, mutants, alleles, parts and fragments of CXCR4 and in particular human CXCR4 that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the immunoglobulin single variable domains and polypeptides of the invention bind to CXCR4 and in particular to human CXCR4. Again, in such a case, the immunoglobulin single variable domains and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e., higher than or lower than), the affinity and specificity with which the immunoglobulin single variable domains of the invention bind to (wild-type) CXCR4, provided a function of CXCR4 is inhibited.
[0171] Inhibition of a function(s) of the first target can be determined by any suitable assay known by the person skilled in the art, such as ELISA, FACS, Scatchard analysis, Alphascreen, SPR, functional assays, etc.
[0172] The efficacy or potency of the immunoglobulin single variable domains and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include ligand displacement assays (Burgess et al., Cancer Res 2006 66:1721-9), dimerization assays (WO2009/007427A2, Goetsch, 2009), signaling assays (Burgess et al., Mol Cancer Ther 9:400-9), proliferation/survival assays (Pacchiana et al., J Biol Chem 2010 September M110.134031), cell adhesion assays (Holt et al., Haematologica 2005 90:479-88) and migration assays (Kong-Beltran et al., Cancer Cell 6:75-84), endothelial cell sprouting assays (Wang et al., J Immunol. 2009; 183:3204-11), and in vivo xenograft models (Jin et al., Cancer Res. 2008 68:4360-8), as well as the assays and animal models used in the experimental part below and in the prior art cited herein. A means to express the inhibition of said first target is by IC50.
[0173] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV has an IC50 of between 200 nM and 1 nM, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nM, for instance determined in a ligand competition assay, a functional cellular assay, such as inhibition of ligand-induced chemotaxis, an Alphascreen assay, etc.
[0174] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV inhibits binding of a natural ligand to said first target, such as e.g. SDF-1 to CXCR4 by about 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or even 100%, e.g. relative to the inhibition in the absence of said first ISV.
[0175] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV inhibits the pharmacologic effect e.g. anaplasia, invasiveness, metastasis, proliferation, differentiation, migration and/or survival, in which said first target is involved by about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or even 100%, e.g. relative to the pharmacologic effect in the absence of said first ISV.
[0176] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV increases apoptosis, cell killing and/or growth arrest of said cell, in which said first target is involved by about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or even 100%, e.g. relative to the increase in the absence of said first ISV.
[0177] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV displaces about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or more of the natural ligand to said first target, e.g. relative to the displacement in the absence of said first ISV.
[0178] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV inhibits signalling by said first target, e.g. kinase activity of said first target, by about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or even 100%, e.g. relative to the inhibition in the absence of said first ISV.
[0179] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV inhibits dimerisation of said first target by about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or even 100%, e.g. relative to the inhibition in the absence of said first ISV.
[0180] Accordingly, the present invention relates to a polypeptide as described herein, wherein said first ISV inhibits chemotaxis by about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95% or even 100% in a chemotaxis assay, e.g. relative to the inhibition in the absence of said first ISV.
[0181] Second Building Block (Anchoring Building Block)
[0182] The second building block, ISV, Nanobody or VHH of the invention has a high affinity for its—the second—target. The second building block, ISV or Nanobody of the invention may for example be directed against an antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of said second target. The second building block, e.g. the second ISV, Nanobody or VHH, is chosen for its high affinity for its target per se, disregarding the influence of any avidity effects.
[0183] Accordingly, the present invention relates to a polypeptide as described herein, wherein said second ISV binds to a second target with an average KD value of between 10 nM and 0.1 pM, such as at an average KD value of 10 nM or less, even more preferably at an average KD value of 9 nM or less, such as less than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as less than 400, 300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, or even less such as less than 0.4 pM. Preferably, the KD is determined by SPR.
[0184] Accordingly, the present invention relates to a polypeptide as described herein, wherein said second ISV has a high affinity when measured as a monovalent.
[0185] Accordingly, the present invention relates to a polypeptide as described herein, wherein said average KD is measured by surface plasmon resonance (SPR) on recombinant protein.
[0186] The present invention also relates to a polypeptide as described herein, wherein said second ISV binds to a second target on the surface of a cell with an EC50 value of between 10 nM and 0.1 pM, such as at an average KD value of 10 nM or less, even more preferably at an average KD value of 9 nM or less, such as less than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as less than 400, 300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, or even less such as less than 0.4 pM.
[0187] Accordingly the present invention relates to a polypeptide as described herein, wherein said average EC50 is measured on cells comprising said target 2 but substantially lacking said target 1.
[0188] Accordingly, the present invention relates to a polypeptide as described herein, wherein said average KD is determined by FACS, Biacore, ELISA, on a monovalent second ISV, such as a Nanobody, or a polypeptide comprising a monovalent second ISV, such as a Nanobody.
[0189] It has been shown in the examples that the KD correlates well with the EC50.
[0190] Said second target can be any target on a cell, e.g. CD123 (OMIM: 308385), provided it is different from said first target. Preferably, said second target is unique to said diseased cell, e.g. a cancer cell. For instance, said second target is not expressed on a normal, healthy cell. However, this will not be the case generally. In most cases, said second target will be present on both normal and diseased cells, e.g. cancer cells. Although the function of said second target might not be vital to said cells, inhibiting its function on normal cells may give rise to some toxicity and side-effects. The present invention further relates to high affinity binders comprised in the polypeptide of the invention which do not or only minimally impair or inhibit the function of normal cells.
[0191] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV binds to an allosteric site of said second target.
[0192] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV does not substantially or only marginally inhibit a function of said second target, e.g. as a monovalent.
[0193] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV has an IC50 of between 100 nM and 10 μM, such as 200 nM, 500 nM, 1 μM or 5 μM, in an Alphascreen assay, competition ELISA, or FACS on cells as e.g., described in the experimental part.
[0194] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV inhibits binding of a natural ligand to said second target by less than about 50%, such as 40%, 30%, or 20% or even less than 10%, e.g. relative to the inhibition in the absence of said second ISV.
[0195] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV inhibits the pharmacologic effect of said second target by less than about 50%, such as 40%, 30%, or 20% or even less than 10%, e.g. relative to the inhibition in the absence of said second ISV.
[0196] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV displaces the natural ligand to said second target by less than about 50%, such as 40%, 30%, or 20% or even less than 10%, e.g. relative to the displacement in the absence of said second ISV.
[0197] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV inhibits signalling by said second target by less than about 50%, such as 40%, 30%, or 20% or even less than 10%, e.g. relative to the inhibition in the absence of said second ISV.
[0198] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV inhibits dimerisation of said first target by less than about 50%, such as 40%, 30%, or 20% or even less than 10%, e.g. relative to the inhibition in the absence of said second ISV.
[0199] Accordingly the present invention relates to a polypeptide as described herein, wherein said second ISV inhibits chemotaxis by less than about 50%, such as 40%, 30%, or 20% or even less than 10% in an chemotaxis assay, e.g. relative to the inhibition in the absence of said second ISV.
Combinations
[0200] In order to increase specificity and thus minimize side-effects and/or toxicity, the second, anchoring target is preferably a tumor-associated antigen (TAA). TAA are typically antigens that are expressed on cells of particular tumors, but that are typically not expressed in normal cells. Often, TAA are antigens that are normally expressed in cells only at particular points in an organism's development (such as during fetal development) and that are being inappropriately expressed in the organism at the present point of development, or are antigens not expressed in normal tissues or cells of an organ now expressing the antigen. Preferred TAA as second, anchoring target include MART-1, carcinoembryonic antigen (“CEA”), gp100, MAGE-1, HER-2, and Lewis.sup.Y antigens.
[0201] Cell surface antigens that are preferentially expressed on AML LSC compared with normal hematopoietic stem cells, and thus preferred as second target, include CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3 and CD25.
[0202] Other tumor-associated antigens suitable as the second target within the polypeptides of the invention include: TAG-72, Ep-CAM, PSMA, PSA, glycolipids such as GD2 and GD3.
[0203] The second, anchoring targets of the invention include also hematopoietic differentiation antigens, i.e. glycoproteins usually associated with cluster differentiation (CD) grouping, such as CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, and CD147; growth factor receptors, including ErbB3 and ErbB4; and Cytokine receptors including Interleukin-2 receptor gamma chain (CD132 antigen); Interleukin-10 receptor alpha chain (IL-10R-A); Interleukin-10 receptor beta chain (IL-10R-B); Interleukin-12 receptor beta-1 chain (IL-12R-beta1); Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2); Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213 aI antigen); Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding protein); Interleukin-17 receptor (IL-17 receptor); Interleukin-176 receptor (IL-1713 receptor); Interleukin 21 receptor precursor (IL-21R); Interleukin-1 receptor, type I (IL-1R-1) (CD121a); Interleukin-1 receptor, type II (IL-1R-beta) (CDw121b); Interleukin-1 receptor antagonist protein (IL-1ra); Interleukin-2 receptor alpha chain (CD25 antigen); Interleukin-2 receptor beta chain (CD122 antigen); Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen)
[0204] Accordingly the present invention relates to a polypeptide as described herein, wherein said second, anchoring target is chosen from the group consisting of MART-1, carcinoembryonic antigen (“CEA”), gp100, MAGE-1, HER-2, and Lewis.sup.Y antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, and CD147; growth factor receptors, including ErbB3 and ErbB4; and Cytokine receptors including Interleukin-2 receptor gamma chain (CD132 antigen); Interleukin-10 receptor alpha chain (IL-10R-A); Interleukin-10 receptor beta chain (IL-10R-B); Interleukin-12 receptor beta-1 chain (IL-12R-beta1); Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2); Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213 aI antigen); Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding protein); Interleukin-17 receptor (IL-17 receptor); Interleukin-176 receptor (IL-17B receptor); Interleukin 21 receptor precursor (IL-21R); Interleukin-1 receptor, type I (IL-1R-1) (CD121a); Interleukin-1 receptor, type II (IL-1R-beta) (CDw121b); Interleukin-1 receptor antagonist protein (IL-1ra); Interleukin-2 receptor alpha chain (CD25 antigen); Interleukin-2 receptor beta chain (CD122 antigen); Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen).
[0205] Accordingly the present invention relates to a polypeptide as described herein 1, wherein said first, functional target is chosen from the group consisting of GPCRs, Receptor Tyrosine Kinases, DDR1, Discoidin I (CD167a antigen), DDR2, ErbB-1, C-erbB-2, FGFR-1, FGFR-3, CD135 antigen, CD 117 antigen, Protein tyrosine kinase-1, c-Met, CD148 antigen, C-ret, ROR1, ROR2, Tie-1, Tie-2, CD202b antigen, Trk-A, Trk-B, Trk-C, VEGFR-1, VEGFR-2, VEGFR-3, Notch receptor 1-4, FAS receptor, DRS, DR4, CD47, CX3CR1, CXCR-3, CXCR-4, CXCR-7, Chemokine binding protein 2, and CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11; and said second target is chosen from the group consisting of MART-1, carcinoembryonic antigen (“CEA”), gp100, MAGE-1, HER-2, and Lewis.sup.Y antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, and CD147; growth factor receptors, including ErbB3 and ErbB4; and Cytokine receptors including Interleukin-2 receptor gamma chain (CD132 antigen); Interleukin-10 receptor alpha chain (IL-10R-A); Interleukin-10 receptor beta chain (IL-10R-B); Interleukin-12 receptor beta-1 chain (IL-12R-beta1); Interleukin-12 receptor beta-2 chain (IL-12 receptor beta-2); Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1) (CD213 aI antigen); Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding protein); Interleukin-17 receptor (IL-17 receptor); Interleukin-17B receptor (IL-17B receptor); Interleukin 21 receptor precursor (IL-21R); Interleukin-1 receptor, type I (IL-1R-1) (CD121a); Interleukin-1 receptor, type II (IL-1R-beta) (CDw121b); Interleukin-1 receptor antagonist protein (IL-1ra); Interleukin-2 receptor alpha chain (CD25 antigen); Interleukin-2 receptor beta chain (CD122 antigen); Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen).
[0206] As used herein “epidermal growth factor receptor” (EGFR, ErbB1, HER1) refers to naturally occurring or endogenous mammalian EGFR proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian EGFR protein (e.g., recombinant proteins, synthetic proteins (i.e., produced using the methods of synthetic organic chemistry)). Accordingly, as defined herein, the term includes mature EGFR protein, polymorphic or allelic variants, and other isoforms of an EGFR (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g., lipidated, glycosylated). Naturally occurring or endogenous EGFR include wild type proteins such as mature EGFR, polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be recovered or isolated from a source which naturally produces EGFR, for example. These proteins and proteins having the same amino acid sequence as a naturally occurring or endogenous corresponding EGFR, are referred to by the name of the corresponding mammal. For example, where the corresponding mammal is a human, the protein is designated as a human EGFR. An ISV (e.g., Nanobody) that inhibits binding of EGF and/or TGF alpha to EGFR inhibits binding in the EGFR binding assay or EGFR kinase assay described herein with an IC50 of about 1 [mu]M or less, about 500 nM or less, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 10 nM or less or about 1 nM or less.
[0207] Accordingly the present invention relates to a polypeptide as described herein, wherein said first target (functional target) and said second target (anchoring target) are chosen from the group consisting of
TABLE-US-00001 functional target anchoring target RTK TAA GPCR TAA CXCR4 (OMIM: 162643) CD123 (OMIM: 308385) DR5 (OMIM: 603612) EpCam (OMIM: 185535) DR4 (OMIM: 126452) EpCam (OMIM: 185535) CD95 (OMIM: 134637) EpCam (OMIM: 185535) CD47 (OMIM: 601028) CD123 (OMIM: 308385) CD47 (OMIM: 601028) EpCam (OMIM: 185535) EGFR (OMIM: 131550) CEA (OMIM: 114890) CXCR4 (OMIM: 162643) CD4 (OMIM/186940) IL12Rβ1 (OMIM: 601604) CD4 (OMIM/186940) IL12Rβ2 (OMIM: 601642) CD4 (OMIM/186940) IL23R (OMIM: 605580) CD4 (OMIM/186940)
[0208] In particular, the present invention relates to a polypeptide according to the invention, wherein said first target and said second target are chosen from the group consisting of: [0209] Receptor Tyrosine Kinase as a first target and a tumor-associated antigen (TAA) as a second target; [0210] G-Protein-Coupled Receptor (GPCR) as a first target and a hematopoietic differentiation antigen as a second target; [0211] Receptor Tyrosine Kinase as a first target and a hematopoietic differentiation antigen as a second target; [0212] G-Protein-Coupled Receptor (GPCR) as a first target and a tumor-associated antigen (TAA) as a second target; [0213] CXCR4 as a first target and CD123 as a second target; [0214] DR5 as first target and EpCam as a second target; [0215] DR4 as first target and EpCam as a second target; [0216] CD95 as first target and EpCam as a second target; [0217] CD47as first target and CD123 as a second target; [0218] CD47 as first target and EpCam as a second target; [0219] EGFR as first target and CEA as a second target [0220] CD4 as first target and CXCR4 as a second target [0221] IL12Rβ1 as first target and CD4 as a second target [0222] IL12Rβ2 as first target and CD4 as a second target, and [0223] IL23R as first target and CD4 as a second target
[0224] The present inventors have also demonstrated that a first target can become a second target and vice versa, depending on the affinity and the functional properties of the respective ISVs (see e.g. ISVs binding CXCR4).
[0225] The present inventors further demonstrated that the absolute copy number of the first and second target, but also the ratio of the first target and second target, on the cell surface can be a determinant in the specificity of the eventual binding, and thus in the toxicity and/or side effects. Preferably, a low number of copies is present of said first, functional target. Preferably, a high number of copies is present of said second, anchoring target. Even more preferably, a low ratio of the first, functional target and second, anchoring target is present on the cell surface number.
[0226] Accordingly the present invention relates to a polypeptide as described herein, wherein said cell comprises between 1,000 and 40,000 copies, such as between 10,000 and 20,000 copies of said first target on the surface of said cell.
[0227] Accordingly the present invention relates to a polypeptide as described herein, wherein said cell comprises between 40,000 and 100,000 copies, such as between 60,000 and 80,000 copies of said second target on the surface of said cell.
[0228] Accordingly the present invention relates to a polypeptide as described herein, wherein said cell comprises a ratio of 0.01 to 0.9 of said first, functional target and said second, anchoring target, even more preferably between 0.2 to 0.8, 0.3 to 0.7, 0.4 to 0.6, such as a ratio of 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, preferably a ratio of 0.5.
[0229] As such, the polypeptides and compositions of the present invention can be used for the diagnosis, prevention and treatment of diseases and disorders of the present invention (herein also “diseases and disorders of the present invention”) which include, but are not limited to cancer. The term “cancer” refers to the pathological condition in mammals that is typically characterized by dysregulated cellular proliferation or survival. Examples of cancer include, but are not limited to, carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas: breast cancer, ovarian cancer, cervical cancer, glioblastoma, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma (see e.g., Cancer, Principles and practice (DeVita, V. T. et al. eds 1997) for additional cancers); as well as any metastasis of any of the above cancers, as well as non-cancer indications such as nasal polyposis; as well as other disorders and diseases described herein. In particular, the polypeptides and compositions of the present invention can be used for the diagnosis, prevention and treatment of diseases involving EGFR mediated metastasis, chemotaxis, cell adhesion, trans endothelial migration, cell proliferation and/or survival. Cancers characterized by expression of EGFR on the surface of cancerous cells (EGFR-expressing cancers) include, for example, bladder cancer, ovarian cancer, colorectal cancer, breast cancer, lung cancer (e.g., non-small cell lung carcinoma), gastric cancer, pancreatic cancer, prostate cancer, head and neck cancer, renal cancer and gall bladder cancer.
[0230] For a general description of immunoglobulin single variable domains, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly describes immunoglobulin single variable domains of the so-called “V.sub.H3 class” (i.e., immunoglobulin single variable domains with a high degree of sequence homology to human germline sequences of the V.sub.H3 class such as DP-47, DP-51 or DP-29), which form a preferred aspect of this invention. It should, however, be noted that the invention in its broadest sense generally covers any type of immunoglobulin single variable domains and for example also covers the immunoglobulin single variable domains belonging to the so-called “V.sub.H4 class” (i.e., immunoglobulin single variable domains with a high degree of sequence homology to human germline sequences of the V.sub.H4 class such as DP-78), as for example described in WO 07/118670.
[0231] Generally, immunoglobulin single variable domains (in particular V.sub.HH sequences and sequence optimized immunoglobulin single variable domains) can in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences (again as further described herein).
[0232] Thus, generally, an immunoglobulin single variable domain can be defined as an amino acid sequence with the (general) structure (cf. formula 1 below)
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively.
[0233] In a preferred aspect, the invention provides polypeptides comprising at least an immunoglobulin single variable domain that is an amino acid sequence with the (general) structure
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: [0234] i) at least one of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-1 below; and in which: [0235] ii) said amino acid sequence has at least 80%, more preferably 90%, even more preferably 95% amino acid identity with at least one of the immunoglobulin single variable domains as shown in WO 2009/138519 (see SEQ ID NOs: 1 to 125 in WO 2009/138519), in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences) are disregarded; and in which: [0236] iii) the CDR sequences are generally as further defined herein (e.g., the CDR1, CDR2 and CDR3 in a combination as can be determined with the information provided herein, noting that the CDR definitions are calculated according to the Kabat numbering system).
TABLE-US-00002 TABLE A-1 Hallmark Residues in VHHs Human Position V.sub.H3 Hallmark Residues 11 L, V; L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; predomi- preferably L nantly L 37 V, I, F; F.sup.(1), Y, V, L, A, H, S, I, W, C, N, G, D, T, P, usually V preferably F.sup.(1) or Y 44.sup.(8) G E.sup.(3), Q.sup.(3), G.sup.(2), D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G.sup.(2), E.sup.(3) or Q.sup.(3); most preferably G.sup.(2) or Q.sup.(3). 45.sup.(8) L L.sup.(2), R.sup.(3), P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L.sup.(2) or R.sup.(3) 47.sup.(8) W, Y F.sup.(1), L.sup.(1) or W.sup.(2) G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W.sup.(2), L.sup.(1) or F.sup.(1) 83 R or K; R, K.sup.(5), T, E.sup.(5), Q, N, S, I, V, G, M, L, A, D, Y, H; usually R preferably K or R; most preferably K 84 A, T, D; P.sup.(5), S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; predomi- preferably P nantly A 103 W W.sup.(4), R.sup.(6), G, S, K, A, M, Y, L, F, T, N, V, Q, P.sup.(6), E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108 L, M or T; Q, L.sup.(7), R, P, E, K, S, T, M, A, H; preferably Q or predomi- L.sup.(7) nantly L Notes: .sup.(1)In particular, but not exclusively, in combination with KERE or KQRE at positions 43-46. .sup.(2)Usually as GLEW at positions 44-47. .sup.(3)Usually as KERE or KQRE at positions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47. Alternatively, also sequences such as TERE (for example TEREL), TQRE (for example TQREL), KECE (for example KECEL or KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible. Some other possible, but less preferred sequences include for example DECKL and NVCEL. .sup.(4)With both GLEW at positions 44-47 and KERE or KQRE at positions 43-46. .sup.(5)Often as KP or EP at positions 83-84 of naturally occurring V.sub.HH domains. .sup.(6)In particular, but not exclusively, in combination with GLEW at positions 44-47. .sup.(7)With the proviso that when positions 44-47 are GLEW, position 108 is always Q in (non-humanized) V.sub.HH sequences that also contain a W at 103. .sup.(8)The GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.
[0237] Again, such immunoglobulin single variable domains may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V.sub.HH sequences (i.e., from a suitable species of Camelid, e.g., llama) or synthetic or semi-synthetic VHs or VLs (e.g., from human). Such immunoglobulin single variable domains may include “humanized” or otherwise “sequence optimized” VHHs, “camelized” immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences, i.e., camelized VHs), as well as human VHs, human VLs, camelid VHHs that have been altered by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. As mentioned herein, a particularly preferred class of immunoglobulin single variable domains of the invention comprises immunoglobulin single variable domains with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V.sub.HH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V.sub.HH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V.sub.H domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V.sub.HH domain as a starting material.
[0238] Another particularly preferred class of immunoglobulin single variable domains of the invention comprises immunoglobulin single variable domains with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V.sub.H domain, but that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V.sub.H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V.sub.HH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the description herein. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the V.sub.H-V.sub.L interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see also for example WO 94/04678 and Davies and Riechmann (1994 and 1996)). Preferably, the V.sub.H sequence that is used as a starting material or starting point for generating or designing the camelized immunoglobulin single variable domains is preferably a V.sub.H sequence from a mammal, more preferably the V.sub.H sequence of a human being, such as a V.sub.H3 sequence. However, it should be noted that such camelized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V.sub.H domain as a starting material.
[0239] For example, again as further described herein, both “humanization” and “camelization” can be performed by providing a nucleotide sequence that encodes a naturally occurring V.sub.HH domain or V.sub.H domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” immunoglobulin single variable domains of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired immunoglobulin single variable domains of the invention. Alternatively, based on the amino acid sequence of a naturally occurring V.sub.HH domain or V.sub.H domain, respectively, the amino acid sequence of the desired humanized or camelized immunoglobulin single variable domains of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V.sub.HH domain or V.sub.H domain, respectively, a nucleotide sequence encoding the desired humanized or camelized immunoglobulin single variable domains of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired immunoglobulin single variable domains of the invention.
[0240] Generally, proteins or polypeptides that comprise or essentially consist of a single building block, single immunoglobulin single variable domain or single Nanobody will be referred to herein as “monovalent” proteins or polypeptides or as “monovalent constructs”, or as monovalent building block, monovalent immunoglobulin single variable domain or monovalent Nanobody, respectively. Proteins and polypeptides that comprise or essentially consist of two or more immunoglobulin single variable domains (such as at least two immunoglobulin single variable domains of the invention) will be referred to herein as “multivalent” proteins or polypeptides or as “multivalent constructs”, and these provide certain advantages compared to the corresponding monovalent immunoglobulin single variable domains of the invention. Some non-limiting examples of such multivalent constructs will become clear from the further description herein. The polypeptides of the invention are “multivalent”, i.e. comprising two or more building blocks or ISVs of which at least the first building block, ISV or Nanobody and the second building block, ISV or Nanobody are different, and directed against different targets, such as antigens or antigenic determinants. Polypeptides of the invention that contain at least two building blocks, ISVs or Nanobodies, in which at least one building block, ISV or Nanobody is directed against a first antigen (i.e., against the first target, such as e.g. CXCR4) and at least one building block, ISV or Nanobody is directed against a second antigen (i.e., against the second target which is different from the first target, such as e.g. CD123), will also be referred to as “multispecific” polypeptides of the invention, and the building blocks, ISVs or Nanobodies present in such polypeptides will also be referred to herein as being in a “multivalent format”. Thus, for example, a “bispecific” polypeptide of the invention is a polypeptide that comprises at least one building block, ISV or Nanobody directed against a first target (e.g. CXCR4) and at least one further building block, ISV or Nanobody directed against a second target (i.e., directed against a second target different from said first target, e.g. CD123), whereas a “trispecific” polypeptide of the invention is a polypeptide that comprises at least one building block, ISV or Nanobody directed against a first target (e.g., CXCR4), a second building block, ISV or Nanobody directed against a second target different from said first target (e.g. CD123) and at least one further building block, ISV or Nanobody directed against a third antigen (i.e., different from both the first and the second target), such as, for instance, serum albumin; etc. As will be clear from the description, the invention is not limited to bispecific polypeptides, in the sense that a multispecific polypeptide of the invention may comprise at least a first building block, ISV or Nanobody against a first target, a second building block, ISV or Nanobody against a second target and any number of building blocks, ISVs or Nanobodies directed against one or more targets, which may be the same or different from the first and/or second target, respectively. The building blocks, ISVs or Nanobodies can optionally be linked via linker sequences.
[0241] Accordingly, the present invention also relates to a trispecific or multispecific polypeptide, comprising or essentially consisting of at least three binding moieties, such as three ISVs, wherein at least one of said at least three binding moieties is directed against a first target with a low affinity, at least one of said at least three binding moieties is directed against a second target with a high affinity and at least a third binding moiety increasing half life, such as e.g. an Albumin binder.
[0242] As will be clear from the further description above and herein, the immunoglobulin single variable domains of the invention can be used as “building blocks” to form polypeptides of the invention, e.g., by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as, without limitations, the bi-/tri-/tetra-/multivalent and bi-/tri-/tetra-/multispecific polypeptides of the invention described herein) which combine within one molecule one or more desired properties or biological functions.
[0243] The compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more immunoglobulin single variable domains of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.
[0244] The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as “formatting” said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be “formatted” or to be “in the format of” said compound or polypeptide of the invention. Examples of ways in which an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted immunoglobulin single variable domains form a further aspect of the invention.
[0245] For example, such further groups, residues, moieties or binding units may be one or more additional immunoglobulin single variable domains, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, immunoglobulin single variable domains that are suitable for use as a domain antibody, single domain antibodies, immunoglobulin single variable domains (ISVs) that are suitable for use as a single domain antibody, “dAb”'s, immunoglobulin single variable domains that are suitable for use as a dAb, or Nanobodies. Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more immunoglobulin single variable domains of the invention so as to provide a “derivative” of an amino acid sequence or polypeptide of the invention, as further described herein.
[0246] Also within the scope of the present invention are compounds or constructs, which comprise or essentially consist of one or more derivatives as described herein, and optionally further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are immunoglobulin single variable domains. In the compounds or constructs described above, the one or more immunoglobulin single variable domains of the invention and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are immunoglobulin single variable domains, the linkers may also be immunoglobulin single variable domains, so that the resulting compound or construct is a fusion protein or fusion polypeptide.
[0247] In a specific, but non-limiting aspect of the invention, which will be further described herein, the polypeptides of the invention have an increased half-life in serum (as further described herein) compared to the immunoglobulin single variable domain from which they have been derived. For example, an immunoglobulin single variable domain of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the invention with increased half-life.
[0248] In a specific aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise immunoglobulin single variable domains or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); immunoglobulin single variable domains of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention which comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) which increases the half-life of the amino acid sequence of the invention. Examples of polypeptides of the invention which comprise such half-life extending moieties or immunoglobulin single variable domains will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more immunoglobulin single variable domains of the invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, immunoglobulin single variable domains that are suitable for use as a domain antibody, single domain antibodies, immunoglobulin single variable domains that are suitable for use as a single domain antibody, “dAb”'s, immunoglobulin single variable domains that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to the further description and references mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more immunoglobulin single variable domains of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins, such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489, WO2008/068280, WO2009/127691 and PCT/EP2011/051559.
[0249] Generally, the compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds or polypeptides of the invention with increased half-life may have a half-life e.g., in humans that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.
[0250] In a preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention have a serum half-life e.g. in humans that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.
[0251] In another preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
[0252] In a particularly preferred but non-limiting aspect of the invention, the invention provides a polypeptide of the invention comprising a first and a second immunoglobulin single variable domain (ISV), wherein said first ISV binds to a first target on the surface of a cell with a low affinity and when bound inhibits a function of said first target; and said second ISV binds to a second target on the surface of said cell with a high affinity, and preferably inhibits a function of said second target minimally, wherein said first target is different from said second target; and further comprising one or more (preferably one) serum albumin binding immunoglobulin single variable domain as described herein, e.g. the serum albumin binding immunoglobulin single variable domain of SEQ ID NO: 114 or 115 (Table B-4).
[0253] Polypeptide-Drug Conjugates (PDCs)
[0254] In some embodiments, the polypeptides of the invention are conjugated with drugs to form polypeptide-drug conjugates (PDCs). Contemporaneous antibody-drug conjugates (ADCs) are used in oncology applications, where the use of antibody-drug conjugates for the local delivery of drugs, such as cytotoxic or cytostatic agents, toxin or toxin, moieties, allows for the targeted delivery of the drug moiety to tumors, which can allow higher efficacy, lower toxicity, etc. These ADCs have three components: (1) a monoclonal antibody conjugated through a (2) linker to a (3) toxin moiety or toxin. An overview of this technology is provided in Ducry et al., Bioconjugate Chem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) and Senter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which are hereby incorporated by reference in their entirety. The PDCs also have three components: (1) a polypeptide conjugated through a (2) linker to a (3) drug, such as a toxin moiety or toxin. The person skilled in the art will appreciate that the technology, methods, means, etc. of ADCs are equally applicable to PDCs.
[0255] The invention provides polypeptides of the invention comprising a drug, such as a toxin or toxin moiety.
[0256] The drug, e.g. toxin moiety or toxin can be linked or conjugated to the polypeptide using any suitable method. Generally, conjugation is done by covalent attachment to the polypeptide, as known in the art, and generally relies on a linker, often a peptide linkage. For example, the drug, such as toxin moiety or toxin can be covalently bonded to the polypeptide directly or through a suitable linker. Suitable linkers can include noncleavable or cleavable linkers, for example, pH cleavable linkers that comprise a cleavage site for a cellular enzyme (e.g., cellular esterases, cellular proteases such as cathepsin B). Such cleavable linkers can be used to prepare a ligand that can release a drug, such as a toxin moiety or toxin after the polypeptide is internalized. As will be appreciated by those in the art, the number of drug moieties per polypeptide can change, depending on the conditions of the reaction, and can vary from 1:1 to 10:1 drug:polypeptide. As will also be appreciated by those in the art, the actual number is an average. A variety of methods for linking or conjugating a drug, such as a toxin moiety or toxin to a polypeptide can be used. The particular method selected will depend on the drug, such as a toxin moiety or toxin and polypeptide to be linked or conjugated. If desired, linkers that contain terminal functional groups can be used to link the polypeptide and drug, e.g. a toxin moiety or toxin. Generally, conjugation is accomplished by reacting the drug, e.g. a toxin moiety or toxin that contains a reactive functional group (or is modified to contain a reactive functional group) with a linker or directly with a polypeptide. Covalent bonds formed by reacting a drug, e.g. a toxin moiety or toxin that contains (or is modified to contain) a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond. If desired, a suitable reactive chemical group can be added to polypeptide or to a linker using any suitable method. (See, e.g., Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).) Many suitable reactive chemical group combinations are known in the art, for example an amine group can react with an electrophilic group such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Thiols can react with maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages. Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)).
[0257] As described below, the drug of the PDC can be any number of agents, including but not limited to cytostatic agents, cytotoxic agents such as chemotherapeutic agents, growth inhibitory agents, toxins (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), toxin moieties, or a radioactive isotope (that is, a radioconjugate) are provided. In other embodiments, the invention further provides methods of using the PDCs.
[0258] Drugs for use in the present invention include cytotoxic drugs, particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs. Exemplary classes of cytotoxic agents include the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols.
[0259] Members of these classes include, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, maytansinoids (including DM1), monomethyl-auristatin E (MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and their analogues.
[0260] Drugs, such as toxins may be used as polypeptides-toxin conjugates and include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
[0261] Conjugates of a polypeptide of the invention and one or more small molecule toxins, such as a maytansinoids, dolastatins, auristatins, a trichothecene, calicheamicin, and CC1065, and the derivatives of these toxins that have toxin activity, are contemplated.
[0262] Other drugs, such as antitumor agents that can be conjugated to the polypeptides of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).
[0263] Drugs, such as enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
[0264] The present invention further contemplates a PDC formed between a polypeptide of the invention and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
[0265] For selective destruction of the tumor, the polypeptide of the invention may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.
[0266] The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as Tc99m or 1123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail.
[0267] The generation of polypeptide-drug conjugate compounds can be accomplished by any technique known to the skilled artisan in the field of ADCs. Briefly, the polypeptide-drug conjugate compounds can include polypeptide of the invention as the Antibody unit, a drug, and optionally a linker that joins the drug and the binding agent.
[0268] Methods of determining whether a drug or an antibody-drug conjugate exerts an effect, e.g. a cytostatic and/or cytotoxic effect on a cell are known. Generally, the effect, e.g. a cytotoxic or cytostatic activity of an Antibody Drug Conjugate can be measured by: exposing mammalian cells expressing a target protein of the Antibody Drug Conjugate in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the Antibody Drug Conjugate. These methods are equally applicable to PDCs.
[0269] Accordingly the invention relates to a polypeptide of the invention further comprising a drug, such as a toxin or toxin moiety.
[0270] Accordingly, the present invention relates to a polypeptide according to the invention conjugated to a drug, such as a toxin or toxin moiety.
[0271] In view of the specificity, the polypeptides of the invention are also very suitable for conjugation to imaging agents. Suitable imaging agents for conjugating to antibodies are well known in the art, and similarly useful for conjugating to the polypeptides of the present invention. Suitable imaging agents include but are not limited to molecules preferably selected from the group consisting of organic molecules, enzyme labels, radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, digoxigenin, biotin, metal complexes, metals, colloidal gold, fluorescent label, metallic label, biotin, chemiluminescent, bioluminescent, chromophore and mixtures thereof.
[0272] Accordingly, the present invention relates to a polypeptide according to the invention, further comprising an imaging agent, including, but not limited to a molecule preferably selected from the group consisting of organic molecules, enzyme labels, radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, digoxigenin, biotin, metal complexes, metals, colloidal gold, fluorescent label, metallic label, biotin, chemiluminescent, bioluminescent, chromophore and mixtures thereof.
[0273] Linkers
[0274] In the polypeptides of the invention, the two or more building blocks, ISVs or Nanobodies and the optionally one or more polypeptides one or more other groups, drugs, agents, residues, moieties or binding units may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.
[0275] Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.
[0276] Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V.sub.H and V.sub.L domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody by itself forms a complete antigen-binding site).
[0277] For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly.sub.xser.sub.y).sub.z, such as (for example (gly.sub.4ser).sub.3 or (gly.sub.3ser.sub.2).sub.3, as described in WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678). Preferred linkers are depicted in Table B-5.
[0278] Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).
[0279] Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.
[0280] It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for a chemokine, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[0281] For example, in multivalent polypeptides of the invention that comprise building blocks, ISVs or Nanobodies directed against a first and second target, the length and flexibility of the linker are preferably such that it allows each building block, ISV or Nanobody of the invention present in the polypeptide to bind to its cognate target, e.g. the antigenic determinant on each of the targets. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[0282] It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the Nanobodies of the invention). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[0283] Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[0284] Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more building blocks, ISV or Nanobodies, it is possible to link them by use of a linker with three or more “arms”, which each “arm” being linked to a building block, ISV or Nanobody, so as to provide a “star-shaped” construct. It is also possible, although usually less preferred, to use circular constructs.
[0285] Therapeutic and Diagnostic Compositions and Uses
[0286] The invention provides compositions comprising the polypeptides of the invention, including PDCs of the invention, and a pharmaceutically acceptable carrier, diluent or excipient, and therapeutic and diagnostic methods that employ the polypeptides or compositions of the invention. The polypeptides, including PDCs, according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vivo diagnostic applications and the like. Therapeutic and prophylactic uses of polypeptides, including PDCs, of the invention involve the administration of polypeptides, including PDCs, according to the invention to a recipient mammal, such as a human.
[0287] Substantially pure polypeptides and PDCs of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the polypeptides and PDCs may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).
[0288] For example, the polypeptides and PDCs of the present invention will typically find use in preventing, suppressing or treating disease states. For example, polypeptides or PDCs can be administered to treat, suppress or prevent a chronic inflammatory disease, allergic hypersensitivity, cancer, bacterial or viral infection, autoimmune disorders (which include, but are not limited to, Type I diabetes, asthma, multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, spondylarthropathy {e.g., ankylosing spondylitis), systemic lupus erythematosus, inflammatory bowel disease {e.g., Crohn's disease, ulcerative colitis), Myasthenia gravis and Behcet's syndrome, psoriasis, endometriosis, and abdominal adhesions {e.g., post abdominal surgery). The polypeptides and PDCs are useful for treating infectious diseases in which cells infected with an infectious agent contain higher levels of cell surface EGFR than uninfected cells or that contain one or more cell surface targets that are not present on non-infected cells, such as a protein that is encoded by the infectious agent {e.g., bacteria, virus). The polypeptides and PDCs of the present invention will typically find use in preventing, suppressing or treating a cancer. For example, polypeptides and PDCs can be administered to treat, suppress or prevent cancer, which include, but are not limited to, carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas: breast cancer, ovarian cancer, cervical cancer, glioblastoma, multiple myeloma (including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic myeloma), prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma (see e.g., Cancer, Principles and practice (DeVita, V. T. et al. eds 1997) for additional cancers); as well as any metastasis of any of the above cancers, as well as non-cancer indications such as nasal polyposis; as well as other disorders and diseases described herein.
[0289] In the instant application, the term “prevention” involves administration of the protective composition prior to the induction of the disease. “Suppression” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. “Treatment” involves administration of the protective composition after disease symptoms become manifest. Treatment includes ameliorating symptoms associated with the disease, and also preventing or delaying the onset of the disease and also lessening the severity or frequency of symptoms of the disease.
[0290] Animal model systems which can be used to assess efficacy of the polypeptides and PDCs of the invention in preventing treating or suppressing disease (e.g., cancer) are available. Suitable models of cancer include, for example, xenograft and orthotopic models of human cancers in animal models, such as the SCID-hu myeloma model (Epstein J, and Yaccoby, S., Methods Mol Med. 773:183-90 (2005), Tassone P, et al, Clin Cancer Res. 11:4251-8 (2005)), mouse models of human lung cancer (e.g., Meuwissen R and Berns A, Genes Dev. CHECK:643-64 (2005)), and mouse models of metastatic cancers (e.g., Kubota J Cell Biochem. 56:4-8 (1994)).
[0291] Generally, the present polypeptides and PDCs will be utilized in purified form together with pharmacologically appropriate carriers. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide- or PDC-complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
[0292] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition). A variety of suitable formulations can be used, including extended release formulations.
[0293] The polypeptides and PDCs of the present invention may be used as separately administered compositions or in conjunction with other agents. The polypeptides and PDCs can be administered and or formulated together with one or more additional therapeutic or active agents. When a polypeptide or PDC is administered with an additional therapeutic agent, the polypeptide or PDC can be administered before, simultaneously with or subsequent to administration of the additional agent. Generally, the polypeptide or PDC and additional agent are administered in a manner that provides an overlap of therapeutic effect.
[0294] The polypeptides and PDCs of the invention can be co-administered (e.g., to treat cancer, an inflammatory disease or other disease) with a variety of suitable co-therapeutic agents, including cytokines, analgesics/antipyretics, antiemetics, and chemotherapeutics.
[0295] Thus the invention provides a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a polypeptide or PDC of the invention and a chemotherapeutic agent, wherein the chemotherapeutic agent is administered at a low dose. Generally the amount of chemotherapeutic agent that is co-administered with a polypeptide of the invention is about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less, of the dose of chemotherapeutic agent alone that is normally administered to a patient. Thus, cotherapy is particularly advantageous when the chemotherapeutic agent causes deleterious or undesirable side effects that may be reduced or eliminated at lower doses.
[0296] Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with polypeptides or PDCs of the present invention, or even combinations of polypeptides and PDCs according to the present invention having different specificities, such as polypeptides or PDCs selected using different target antigens or epitopes, whether or not they are pooled prior to administration.
[0297] The route of administration of pharmaceutical compositions according to the invention may be any suitable route, such as any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the polypeptides and PDCs of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, intrathecally, intraarticularly, via the pulmonary route, or also, appropriately, by direct infusion (e.g., with a catheter). The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. Administration can be local (e.g., local delivery to the lung by pulmonary administration,(e.g., intranasal administration) or local injection directly into a tumor) or systemic as indicated.
[0298] The polypeptides and PDCs of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.
[0299] The compositions containing the polypeptides or PDCs can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a “therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's health, but generally range from 0.005 to 5.0 mg of ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present polypeptides and PDCs or cocktails thereof may also be administered in similar or slightly lower dosages, to prevent, inhibit or delay onset of disease {e.g., to sustain remission or quiescence, or to prevent acute phase). The skilled clinician will be able to determine the appropriate dosing interval to treat, suppress or prevent disease. When polypeptides or PDCs are administered to treat, suppress or prevent a disease, it can be administered up to four times per day, twice weekly, once weekly, once every two weeks, once a month, or once every two months, at a dose of, for example, about 10 [mu]g/kg to about 80 mg/kg, about 100 [mu]g/kg to about 80 mg/kg, about 1 mg/kg to about 80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kg to about 60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 10 mg/kg, about 10 [mu]g/kg to about 10 mg/kg, about 10 [mu]g/kg to about 5 mg/kg, about 10 [mu]g/kg to about 2.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg.
[0300] In particular embodiments, the polypeptide and PDC of the invention is administered at a dose that provides saturation of the anchoring target or a desired serum concentration in vivo. The skilled physician can determine appropriate dosing to achieve saturation, for example by titrating the polypeptide and monitoring the amount of free binding sites of said anchoring target expressing cells or the serum concentration of the polypeptide. Therapeutic regiments that involve administering a therapeutic agent to achieve target saturation or a desired serum concentration of agent are common in the art, particularly in the field of oncology.
[0301] Treatment or therapy performed using the compositions described herein is considered “effective” if one or more symptoms are reduced (e.g., by at least 10% or at least one point on a clinical assessment scale), relative to such symptoms present before treatment, or relative to such symptoms in an individual (human or model animal) not treated with such composition or other suitable control. Symptoms will obviously vary depending upon the disease or disorder targeted, but can be measured by an ordinarily skilled clinician or technician. Such symptoms can be measured, for example, by monitoring the level of one or more biochemical indicators of the disease or disorder (e.g., levels of an enzyme or metabolite correlated with the disease, affected cell numbers, etc.), by monitoring physical manifestations (e.g., inflammation, tumor size, etc.), or by an accepted clinical assessment scale. A sustained (e.g., one day or more, preferably longer) reduction in disease or disorder symptoms by at least 10% or by one or more points on a given clinical scale is indicative of “effective” treatment. Similarly, prophylaxis performed using a composition as described herein is “effective” if the onset or severity of one or more symptoms is delayed, reduced or abolished relative to such symptoms in a similar individual (human or animal model) not treated with the composition.
[0302] A composition containing polypeptides and/or PDCs according to the present invention may be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the ligands and selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the ligands, e.g. antibodies, cell-surface receptors or binding proteins thereof whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
[0303] Accordingly, the present invention relates to a pharmaceutical composition comprising a polypeptide or PDC according to the invention.
[0304] Accordingly, the present invention relates to a method for delivering a prophylactic or therapeutic polypeptide, PDC or imaging agent to a specific location, tissue or cell type in the body, the method comprising the steps of administering to a subject a polypeptide according to the invention.
[0305] Accordingly, the present invention relates to a method for treating a subject in need thereof comprising administering a polypeptide or PDC according to the invention.
[0306] Accordingly, the present invention relates to a polypeptide or PDC according to the invention for use in treating a subject in need thereof.
[0307] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Modifications and variation of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
[0308] The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures.
[0309] The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove.
[0310] Experimental Section
EXAMPLE 1
Preferential Targeting of Leukemic Cells with CXCR4-CD123 Bispecific Polypeptides
EXAMPLE 1.1
Experimental Set Up for Designing Bispecific CXCR4 and CD123 Polypeptides
[0311] With the generation of bispecific anti-CXCR4-CD123 Nanobodies we aimed to generate a high affinity and high potency antagonist for CXCR4 on cells that express both the CXCR4 and CD123 receptors, as a model system for cancer cells, but not on cells that express primarily CXCR4, which represent normal cells, all in order to minimize side-effects or toxicity.
[0312] To reach this selectivity, it was hypothesized that the anti-CXCR4 Nanobody on one arm (the functional ISV) needs to be a full antagonist, but with only a low to moderate affinity. The anti-CD123 Nanobody on the other arm serves (the anchoring ISV) to increase the affinity and potency of the anti-CXCR4 Nanobody on cells which co-express both receptors by avidity. Simultaneous binding to 2 membrane receptors will increase the affinity of the bispecific over monovalent Nanobodies. For the CD123 arm, the Nanobody is preferentially a binder, but which does not affect its function, again in order to minimize side-effects or toxicity. Hence, a functional blockade of the CD123 receptor is not required. The model system as set out in
[0313] The affinity of each of the Nanobodies needed to obtain the increased avidity is a priori unknown; when the affinity is too high, the bispecific will also bind to cells that express only one receptor, which is not desired. Thereto we set out to design selection procedures for Nanobodies with different affinities to IL3Rα to be combined with low to moderate potency CXCR4 Nanobodies.
EXAMPLE 1.2
Production of Monovalent Nanobodies
[0314] Monovalent CXCR4 and CD123-specific Nanobodies were produced in E. coli and expressed as C-terminal linked FLAG3, His6-tagged proteins in expression vector pAX129. The amino acid sequences are depicted in Tables 1 and 2 for monovalent CXCR4-building blocks and monovalent CD123-building blocks, respectively. Expression was induced by IPTG and allowed to continue for 4 h at 37° C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets. Nanobodies were purified from these extracts using immobilized metal affinity chromatography (IMAC) and a buffer exchange to D-PBS. Purity and integrity was confirmed by SDS-PAGE.
EXAMPLE 1.3
Characteristics of Anti-CD123 Specific Nanobodies
[0315] In order to minimize potential side-effects and/or toxicity, the anti-CD123 Nanobodies do preferably not affect the function of the IL3Rα, which is also expressed on normal cells. Furthermore, in order to avoid any complication by the potential introduction of epitope diversity, and to ensure that any gain of function/selectivity in the Proof of Concept (PoC) study is defined only by the relative affinity (i.e. the affinity of the monovalent building block), we set out to identify Nanobodies binding to the same epitope but differing only in the relative affinity.
EXAMPLE 1.3.1
Binding of Anti-CD123 Nanobodies to Cells Expressing IL-3Rα
[0316] Nanobody binding to membrane associated human IL-3Rα was analysed on HEK293T cells transfected with pcDNA3.1-IL3Rα (NM_002183.2) and non-transfected cells. Surface expression was confirmed by FACS using IL-3Rα specific antibodies (R&D MAB301 and BD Pharminogen 554528), followed by goat anti-mouse PE (Jackson Immuno Research 115-115-164). Briefly, serial dilutions of Nanobodies were allowed to associate for 30 minutes at 4° C. in FACS buffer (PBS 1×+10% FBS+0.05% azide). Following this, cells were washed by centrifugation and probed with 6.7 nM anti-FLAG for 30 minutes at 4° C., to detect bound Nanobody. Detection was done with anti-M13 for 30 minutes at 4° C. Cells were washed and incubated with TOPRO3 to stain for dead cells, which are then removed during the gating procedure. The cells were then analysed via a BD FACSArray. The results are depicted in
[0317] A clear interaction of the CD123 Nanobodies 55A01 and 57A07 with the Hek-IL-3Rα cells is demonstrated, while the lack of binding to HEK293T-wt cells confirmed the specificity of the Nanobodies for IL-3Rα (data not shown).
[0318] Binding of the CD123 Nanobodies was also assessed on leukemic cells that endogenously express both the IL-3Rα and IL-3Rβ chain, i.e. Molm-13 and THP-1 cells. These cells have a much lower IL-3Rα expression level than the transfected HEK-IL-3Ra cells, and with likely more representative expression levels of the receptor. Due to the lower potency of the clones selected for this project, the binding curves were incomplete with respect to saturation of binding. Binding curves and EC50 values are shown in
[0319] The binding studies confirmed that the Nanobodies are able to bind to IL3Rα but do not disrupt the heterodimeric receptor complex of IL3Rα with the IL3Rβ partner, which fulfils a prerequisite of evading a functional blockade of the CD123 receptor signalling.
EXAMPLE 1.3.2
Affinity Determination of CD123 Nanobodies
[0320] The affinities of CD123 specific Nanobodies were further investigated via Surface Plasmon Resonance (SPR) at ProteOn. Immobilisation of recombinant IL-3Rα ectodomain (Sino Biologicals) was done until 761 RU. The Nanobodies were applied at a highest concentration of 1 μM, followed by a three-fold titration, covering 5 further concentrations ranging from 1 μM to 4.1 nM. These were then applied in a single injection cycle, utilising the ProteOn's specific one-shot kinetics approach for kinetic analysis. Evaluation of the association/dissociation data was performed by fitting a 1:1 interaction model (Langmuir binding model). A number of the clones failed to show saturation at the 1 μM concentration, due to the low affinity of the Nanobodies. For CD123 Nanobodies the obtained K.sub.D values correlated well with the apparent affinities retrieved by cell binding EC50 values (see Table 3).
EXAMPLE 1.3.3
Competition of Anti-CD123 Nanobodies with the Anti-CD123 Antibody 7G3
[0321] The functional high affinity human IL-3 receptor is a heterodimer consisting of a ligand binding α subunit and the β subunit. The β subunit does not bind the ligand IL-3 by itself but is required for the high affinity binding of IL-3 to the heterodimeric receptor complex.
[0322] Ligand displacement on Molm-13 cells could not be assessed, as the biotinylated ligand exerted too low binding. Since the IL-3 has only a low affinity to IL-3Rα in absence of the IL-3Rβ, transfected Hek-IL-3Rα cells could not be used either. To assess epitope information, CD123 Nanobodies were analysed in competition with the IL-3Rα-specific mAb 7G3 for binding to IL-3Rα ectodomain in ELISA. The humanised version of anti-IL-3Rα specific monoclonal antibody 7G3, CSL-360, was previously shown to lack functional efficacy in a Phase I clinical trial.
[0323] Briefly, antibody 7G3 (BD, 554527) was coated at 1 ug/ml and blocked in casein (1%) in solution. Nanobodies and biotinylated-IL-3Rα ectodomain [R&D systems, 301-R3/CF] were added and allowed to reach equilibrium over four hours. The plate was then washed and 7G3 associated IL-3Rα was detected via extravidin peroxidase prior to development and subsequent analysis of absorption at OD.sub.450 nm. IC50 values are shown in Table 3.
[0324] CD123 Nanobodies were tested for their capacity to compete with the 7G3 antibody. Two anti-CD123 Nanobodies, i.e. 55A01 and 57A07, were binding to the same epitope as 7G3, but were having different relative affinities and potencies (see also Table 5). Subsequently, these Nanobodies were used for formatting into bispecific polypeptides with anti-CXCR4 Nanobodies (see Example 1.5)
EXAMPLE 1.4
Characteristics of Anti-CXCR4 Specific Nanobodies
[0325] In the present example, the inventors set out to identify and characterize anti-CXCR4 Nanobodies which on the one hand had a low affinity, and on the other hand still were able to act as functional antagonists. Since it is cumbersome to functionally test Nanobodies, which have low to moderate affinity, in particular the absence of any observed function must be due to the low affinity, but not due to binding to e.g. an irrelevant epitope, the inventors used an unconventional approach which is detailed below.
[0326] First a large series of available anti-CXCR4 Nanobodies were assessed for their capacity to antagonize CXCR4 signalling. In previous studies, functional antagonistic Nanobodies specific for CXCR4 were already identified. The present inventors then turned to family members of the functional antagonists, which had lower affinities.
[0327] Furthermore, the inventors observed that in some cases the position of a Nanobody in a bispecific polypeptide could decrease affinity. Without being bound to any theory, is was hypothesized that this may be due to steric hindrance. Hence, by positioning a Nanobody known to have a moderate affinity and having antagonistic activities, in an “unfavourable” location in the bispecific polypeptide, both the affinity and the functional effect could be decreased. As such, the avidity effect of the second Nanobody on the function of the low affinity anti-CXCR4 Nanobody could be discerned.
EXAMPLE 1.4.1
Identification of Low Affinity CXCR4 Nanobodies
[0328] For the generation of CXCR4-IL-3Ra bispecifics, Nanobodies with low to moderate affinities are needed, which recognise the correct epitope for functional blockade. In previous studies functional antagonistic Nanobodies specific for CXCR4 were identified. However, the primary aim during lead selection and identification procedure in those previous studies was to identify high potency candidates, and not the low affinity clones. As the screening cascade of previous studies was focussed on blockade of ligand binding, this hampered the identification of clones that have the correct epitope but low potencies due to low affinity as required in the present study. In case of CXCR4, which is to be embedded in the cellular membrane for correct conformation, no source of recombinant protein was available to specifically search for the low affinity Nanobodies by off-rate analysis in SPR, as done for the IL-3Ra Nanobodies.
[0329] To overcome this problem, the inventors zoomed in on family members of CXCR4 Nanobodies with proven ligand functional blockade of CXCR4 signalling. Nanobodies 14A02, 14E02 and 14D09 are members of the same family, as defined by a conserved CDR3 region. The high affine family member, CXCR4 Nanobody 14A02, has shown to be a potent antagonist of CXCR4 functionality in different cellular assays, including ligand-induced chemotaxis and inhibition of cAMP induction in CXCR4-expressing cells (Table 4).
EXAMPLE 1.4.2
Binding Analysis of CXCR4 Nanobodies
[0330] Binding of CXCR4 Nanobodies to CXCR4-expressing cells was assessed on different cell lines, to assess EC50 values. For CXCR4 the membrane insertion is needed for proper conformation and functionality of the receptor. Therefore CXCR4 Nanobodies were characterized for binding to viral lipoparticles (VLP; Molecular Integral) expressing CXCR4 versus control lipoparticles in ELISA. To this end VLPs were coated at 0.5 U/well overnight at 4° C. using anti-myc antibodies for detection. Over all different binding assays, Nanobody 14D09 always exerted lower binding affinity than 14A02, as indicated by a shift in EC50 values. The results are depicted in
EXAMPLE 1.4.3
Ligand Displacement of CXCR4 Nanobodies
[0331] CXCR4 Nanobodies were analysed for their ability to compete with the ligand CXCL12 (or SDF-1a) for receptor binding, by displacement of biotinylated SDF-1 on Caki-CXCR4 cells in flow cytometry. To this end serial dilutions of Nanobodies were incubated with 30 nM of biotinylated SDF-1 (R&D Systems Fluorokine kit) on cells, after which ligand binding was visualised using extravidin-PE. The biotin-SDF-1 competitor concentration used in this assay was below the EC50 value obtained in dose-titration, where IC50 values should reflect the Ki.
[0332] This assay confirmed that the difference in apparent affinities between the family members 14A09 and 14A02 translates into similar differences in capacity in ligand competition (
[0333] CXCR4 Nanobodies were analysed in radio-ligand displacement assay on membrane extracts of Hek-CXCR4 cells. The advantage of using the radiolabelled ligand is the increased sensitivity, and the low competitor concentration ensures the determination of Ki values (i.e. the real affinity constant) instead of measuring the IC50 value. This makes it possible to accurately determine the potencies of low affine Nanobodies, even though they may not reach full displacement.
[0334] To this end, membrane extracts of Hek293 cells transfected with CXCR4 were incubated with serial dilutions of purified Nanobodies and 75 pM of [.sup.125I]-CXCL12. Non-specific binding was determined in presence of 100 nM cold SDF-1. As controls full blocking CXCR4 Nanobodies 238D4 and 281A6 were included. The assay was performed three times, and average percentages of SDF-1 inhibition were calculated.
[0335] In
[0336] Table 4 lists the characteristics of CXCR4 Nanobodies of low to moderate affinity, as well as of their respective family members.
EXAMPLE 1.5
Bispecific Polypeptides
[0337] In the present example, the inventors combined the different anti-CXCR4 and anti-CD123 Nanobodies which were identified and characterized in the previous experiments, and of which the characteristics are summarized in Table 5. The resulting bispecific polypeptides were subsequently tested for specificity. In particular, eight constructs were made, which are summarized in Table 6.
EXAMPLE 1.5.1
Cloning, Production and Physical Characterisation
[0338] IL3Rα and CXCR4 Nanobodies were cloned in the production vector pAX138 and expressed as Myc-His6-tagged proteins to construct bispecific polypeptides. All eight combinations of the CXCR4 Nanobodies 14D09 (designated as CXCR4#1) and 281F12 (designated as CXCR4#2) and the IL-3Ra Nanobodies 57A07(designated as CD123#1 and 55A01 (designated as CD123#2) were constructed (see Table 6). The Nanobodies were connected with a flexible, long linker of repetitive (GGGGS).sub.7. Individual Nanobodies were amplified in separate PCR reactions to generate N-terminal fragments and C-terminal fragments using primers containing appropriate restriction-sites. Fragments were sequentially inserted into the pAX138 expression vector for E. coli productions. The correct nucleotide sequence of all constructs was confirmed by sequence analysis (see Table 7, bispecific constructs). Subsequently the correct constructs were recloned into the pAX205 vector for production in Pichia pastoris as Flag3-His6-tagged proteins. Plasmids encoding bispecific constructs were linearized by digestion with restriction enzymes prior to the transformation into P. pastoris strain X-33. Small scale test expressions of P. pastoris transformants were done in to select for the clone with good expression levels. Hereto 4 ml scale expressions were performed of 4 clones of each construct in 24-wells deep well plates. Expression of Nanobodies in the medium was evaluated by SDS-PAGE. Medium fractions were collected and used as starting material for immobilized metal affinity chromatography (IMAC) using Nickel Sepharose™ 6 FF. Nanobodies were eluted from the column with 250 mM imidazole and subsequently desalted on Sephadex G-25 Superfine on the Atoll (AT0002) towards dPBS. The purity and integrity of Nanobodies was verified by SDS-PAGE and western blot using anti-VHH and anti-tag detection.
[0339] Monovalent CXCR4 and IL-3Ra-specific Nanobodies were produced in E. coli and expressed as C-terminal linked FLAG3, His6-tagged proteins in expression vector pAX129 as set out in Example 1.2.
EXAMPLE 1.5.2
Characterisation of CXCR4-IL-3Ra Bispecifics
[0340] To assess if the formatting into bispecific constructs affected the target binding capacity of the individual Nanobodies, the bispecific Nanobodies were analysed for binding to recombinant IL-3Ra (R&D Systems) in ELISA and to CXCR4 viral lipoparticles (Integral Molecular).
EXAMPLE 1.5.3
Leukemic Cell Lines Expressing CXCR4 and II-3Ra
[0341] Leukemic cell lines with different expression levels of CXCR4 and CD123 as well as Jurkat cells were used to assess the binding characteristics of the bispecific CXCR4-IL-3Ra polypeptides and their monovalent counterparts. Target expression was confirmed by FACS analysis with anti-hCXCR4 antibody 12G5 (R&D Systems MAB170) and anti-hIL-3Ra antibody 7G3 (BD Pharmingen, 554527), followed by secondary antibody goat-anti-mouse PE (Jackson Immuno Research).
[0342] The results are depicted in
[0343] MOLM-13 cells and THP-1 cells have different relative expression levels of the CXCR4 and II3Ra, with hIL3Ra expression being higher compared to CXCR4 in Molm-13 than in THP-1 cells. U937 cells express the highest levels of CXCR4 and virtually no IL-3Ra.
EXAMPLE 1.5.4
Binding Analysis of CXCR4-CD123
[0344] Binding of bispecific polypeptides in both orientations was analysed on U937 cells expressing only CXCR4, and MOLM-13 and THP-1 cells expressing both targets at different ratios Representative graphs are shown in
[0345] Molm-13 cells.
[0346] The differences in total fluorescence levels between THP-1 and MOLM-13 cells indicates that also the relative expression levels of the two antigens on the cell appear also to contribute to the binding behaviour of the CXCR4-IL-3Ra bispecific polypeptides (
EXAMPLE 1.5.5
Mixing of Cell Lines Jurkat E6-1 and MOLM-13
[0347] The ability of bispecific polypeptides to preferentially bind a cell that expresses both CXCR4 and CD123, rather than a cell expressing CXCR4 alone was evaluated. To this end, a FACS experiment with a mixed population of double-positive (MOLM-13) and CXCR4-only (Jurkat E6-1) cells was done, mimicking the real-life situation with heterogeneous cell populations. In order to distinguish both cell populations, prior to the incubation with the Nanobodies, MOLM-13 cells were labelled with 0.5 μM CFSE (Molecular Probes, Life Technologies) and Jurkat E6-1 with 0.5 μM PKH26 (Sigma-Aldrich), according to the manufacturer's instructions. After mixing both cell lines in the same well at a 1:1 ratio, they were incubated with 6-fold serial dilutions of the different bispecific polypeptides and corresponding monovalent building blocks. The dose-dependent binding of the Nanobodies was detected via the C-terminal FLAG tag using mouse anti-FLAG (Sigma-Aldrich), followed by anti-mouse IgG-APC (Jackson Immununoresearch) and measure with FACSCanto II (Becton, Dickinson and Company). As a control, Nanobody binding was also assessed on either cell line alone.
[0348] As a consequence of the low affinity of the bispecific polypeptide in the IL3Ra-CXCR4 orientation, no EC50 values could be obtained for these constructs. Therefore a direct comparison between the binding to MOLM-13 (CXCR4+/CD123+) versus Jurkat E6-1 (CXCR4+/CD123−) cells was made at one Nanobody concentration (4.6 nM).
[0349] This may indicate that the affinity of the currently used CXCR4 Nanobodies (i.e. EC50 around 10 nM) may still be too high to obtain a gain in selectivity via bispecific binding. To achieve this preferential binding, the result suggests that the affinity for CXCR4 may even be lower, e.g. to the level of the residual binding of the IL3Ra-CXCR4 constructs.
EXAMPLE 1.5.6
Inhibition of CXCR4-Mediated Chemotaxis
[0350] To verify if bispecific CXCR4-IL3Ra polypeptides show increased affinity and potency on cells expressing both receptors, a CXCR4-dependent functional assay was carried out. To this end SDF-1a dependent chemotaxis on Jurkat E6-1 (CXCR4+/IL3Ra−), and MOLM-13 cells (CXCR4+/IL3Ra+) was performed for direct comparison of cells expressing both or only one receptor. Since the functional blockade is only mediated via CXCR4, avidity by the simultaneous binding of the anti-IL3Ra Nanobody® is expected to translate into increased potency in inhibition of chemotaxis.
[0351] Bispecific polypeptides were analyzed for inhibition of CXCL12-induced chemotaxis on cells endogenously expressing CXCR4. As chemoattractant a concentration of 750 pM SDF-1a was used on 100,000 cells/well for the Jurkat cell line, and 500,000 cells/well for the MOLM-13 cell line. On each plate the corresponding monovalent CXCR4 Nanobody was included as reference, allowing to calculate the fold increase of the bispecific within each plate. As additional control 1:1 mixtures of monovalent Nanobodies were included. Representative graphs of the different constructs during are shown in
[0352] These data show a clear gain in potency in inhibition of CXCR4 function for bispecifics in the CXCR4-IL-3Ra orientation on cells that express both antigens, but not on cells that express only CXCR4. This increase was not observed when a mixture of the two monovalent Nanobodies was used, hence depends on linking of the Nanobodies for simultaneous engagement of the targets. The potency enhancements for bispecific constructs of Nanobody CXCR4#2 on Molm-13 cells were 12-15 fold. There seemed no apparent difference between the two IL-3Ra Nanobodies, suggesting that the 8 nM affinity of the lower building block is already sufficient to serve as anchor. The gain in potency is less remarkable for the bispecific constructs of CXCR4#1 building block, where there is only a minor increase compared to the monovalent Nanobody. The potency of CXCR4#1 is higher than for CXCR4#2 (IC50 of 10 nM vs 84 nM), which may indicate it is too high to see an improvement after formatting into bispecific. Alternatively, it is also possible that this Nanobody binds to a different—less favourable—epitope on CXCR4 which limits the formatting potential.
[0353] Representative graphs of the different constructs are shown in
[0354] These data show that bispecifics show a gain in potencies, improving the potency of the CXCR4 Nanobodies to inhibit SDF-1 induced chemotaxis of MOLM-13 cells up to 12-15 fold.
EXAMPLE 2
Preferential Targeting of T Cells with CD4-CXCR4 Bispecific Polypeptides
EXAMPLE 2.1
Characteristics of Monovalent Nanobodies for Formatting
[0355] A panel of CD4 Nanobodies was previously identified from immune libraries with human peripheral blood lymphocytes. Besides its role on T cells, CD4 also serves as primary receptor for HIV1 entry.
[0356] Therefore a panel of CD4 Nanobodies was analysed for the capacity to block the interaction with the viral gp120 protein. Briefly, CD4 Nanobodies were analysed for the ability to compete with gp120 protein binding to recombinant CD4 in ELISA. Briefly, plates were coated with 20 ug/mL sheep anti-gp120 antibodies. lug/mL of HIV1 gp120 protein was captured for 1 hr at room temperature. Biotinylated recombinant human CD4 (Invitrogen) at 0.5 μg/mL was pre-incubated with 500 nM anti-CD4 Nanobodies, or control antibodies mouse anti-CD4 mAb B-A1 and F5 (Diaclone) and rabbit anti-CD4 pAb (ImmunoDiagnostic Inc) for 1hr, after which mixture was transferred to the coated plates and incubated for 1 hr. Detection of bound CD4 was done with Extravidin-peroxidase conjugate.
TABLE-US-00003 TABLE 2.1 Characteristics of monovalent CD4 Nanobody. FACS binding HIV-1 neutralization MOLM-13 THP-1 T cells PMBCs + NL4.3 Nanobody ID EC50 (nM) EC50 (nM) EC50 (nM) IC50 (nM) CD4#8 3F11 0.7 1.0 0.76 29.3
EXAMPLE 2.2
Construction of Bispecific CXCR4-CD4 Polypeptides
[0357] Constructs of the anti-CD4 Nanobody 3F11, designated as CD4#8, and anti-CXCR4 Nanobody 282F12, designated as CXCR4#2, were cloned in the production vector pAX100. This vector is derived from pUC119 and contains a LacZ promoter, a kanamycin resistance gene, a multiple cloning site, an OmpA leader sequence, a C-terminal c-myc tag and a (His)6 tag. Since both targets act as co-receptors for HIV-1 entry, they are expected to be in close proximity on the cell surface. For this reason bispecific polypeptides were generated with flexible spacers of different lengths for linking the two Nanobody building blocks: (Gly.sub.4SerGly.sub.4) (9GS), (Gly.sub.4Ser).sub.5 (25GS), and (Gly.sub.4Ser).sub.7 (35GS), respectively. Bispecific constructs were generated in both orientations, yielding 8 different bispecific constructs (Table 2.2). The correct nucleotide sequence of all constructs was confirmed by sequence analysis (see Table 10 for an overview of all sequences). Subsequently, the correct Nanobody constructs were recloned into the pAX205 vector for production in the yeast Pichia pastoris as FLAG3-His6-tagged proteins, as described in Example 1.2.
TABLE-US-00004 TABLE 2.2 Panel of CXCR4-CD4 Nanobodies CD4#8-CXCR4#2 03F11-9GS-281F12 03F11-25GS-281F12 03F11-35GS-281F12 CXCR4#2-CD4#8 281F12-9GS-03F11 281F12-25GS-03F11 281F12-35GS-03F11
EXAMPLE 2.3
Binding Analysis of Bispecific CXCR4-CD4 Polypeptides
[0358] To assess if the formatting into bispecific constructs affected the binding of the CXCR4#2 Nanobody to CXCR4, the entire set of bispecific polypeptides was analysed for binding to CXCR4 on viral lipoparticles (Integral Molecular). Briefly 2 units of null VLPs and hCXCR4 VLPs were coated on maxisorp plates overnight at 4° C. In the next day free binding sites were blocked using 4% marvel skimmed milk in PBS for 2 h at room temperature. Then, after washing the plate 3× with PBS, 100 nM, 10 nM, 1 nM and 0 nM of purified polypeptides were added to the coated wells and incubated for 1 h at room temperature. After washing 3× with PBS, bound polypeptides were detected with mouse anti-c-myc (Roche, cat#11667149001) and rabbit anti-Mouse-HRP (DAKO, cat#P0260) antibodies both for 1 h at room temperature. Binding was determined based on O.D. values and compared to controls: an irrelevant Nanobody, a non-coated well, both parental monovalent building blocks and a monoclonal anti CXCR4 antibody from R&D (clone:12G5, cat#MAB170).
[0359] The panel of CXCR4-CD4 bispecific polypeptides was analysed for dose-dependent binding to cell lines with different relative expression levels of the two targets in flow cytometry. Cells were incubated with Fc-blocking solution (Miltenyi Biotec cat#130-059-901) for 30 minutes before staining with monoclonal anti-CXCR4 antibody 12G5 (R&D #MAB170) and monoclonal anti-CD4 antibody BA1 (Diaclone #854030000). Bound polypeptides were detected with mouse anti-c-myc (AbD Serotec, cat#MCA2200) and Goat anti-Mouse-PE (Jackson Immunoreseach, cat#115-115-171) antibodies both for 30 min shaking at 4° C. Binding was determined based on MCF values and compared to controls.
[0360] Expression levels of CD4 and CXCR4 on Jurkat cells, THP-1 cells and Molm-13 cells are depicted in
[0361] On Jurkat cells, the CXCR4-CD4 Nanobodies have similar EC50 values as monovalent CXCR4#2, in line with the high CXCR4 expression levels. Nanobodies have a slightly higher fluorescence level than monovalent CXCR4 Nanobodies. On double-positive THP1 cells, a clear shift in the curves of the CXCR4-CD4 bispecific Nanobodies is observed compared to both monovalents, and bispecifics reach much higher plateau levels. The difference in EC50 values between bispecifics and monovalents however is only moderate (0.67 nM vs 1.0 nM vs). On MOLM-13 cells the EC50 value of the bispecifics is similar to that of CD4#8, and also here increased plateau levels are observed. The binding curves of the inverse orientation, CD4-CXCR4 bispecifics are overlapping with the monovalent CD4#8 Nanobody.
[0362] This increase in total fluorescence in flow cytomety may represent additive binding (binding to each target alone), as well as simultaneous binding to both targets on the cell surface, but cell binding assays do not allow to discriminate between these binding modes.
EXAMPLE 2.4
Inhibition of CXCR4-Mediated Chemotaxis by CXCR4-CD4 Bispecifics
[0363] To verify if bispecific CXCR4-IL-3Ra polypeptides show increased affinity and potency on cells expressing both receptors, a CXCR4-dependent functional assay was done. Since MOLM-13 cells express CD4 in conjunction with CXCR4 and CD123, the same experimental set-up was used as described for the CXCR4-CD123 bispecific Nanobodies (see: Example 1.5.5).
[0364] Dose-dependent inhibition of CXCL12-induced chemotaxis by the panel of bispecific CD4-CXCR4 Nanobodies was determined on Jurkat (CXCR4+/CD4 low), and Molm-13 cells (CXCR4++/CD4++). As reference anti-CXCR4 antibody 12G5 was included on each plate. Results of a representative example are shown in
TABLE-US-00005 TABLE 2.3 Blockade of SDF-1 mediated chemotaxis by bispecific CXCR4-CD4 polypeptides. CXCR4.sup.+/CD4.sup.+ CXCR4.sup.+/CD4.sup.low MOLM-13 cells Jurkat E6-1 cells Nanobody Binding Chemotaxis Fold Binding Chemotaxis Fold Nb1 Nb2 EC50 (nM) IC50 (nM) increase EC50 (nM) IC50 (nM) increase CXCR4#2 5.2 86.0 — 7.0 84.2 — CXCR4#2 CD4#8 1.1 0.59 146 11 110 0.8 CD4#8 CXCR4#2 0.7 1.29 67 1.1 460 0.2 CD4#8 0.6 — — 61 — —
EXAMPLE 2.5
CXCR4 Specificity in HIV1 Infection Assays
[0365] Besides its physiological role as homeostatic chemokine receptor, CXCR4 is also used as co-receptor for T-lymphotrophic HIV strains. For entry of the host cell, the viral gp120 protein interacts with CD4 and a co-receptor, which can be either CCR5 or CXCR4. HIV1 strains can be either dependent on CCR5 usage (R5), on CXCR4 usage (X4), or can be dual-tropic, being able to use either receptor for entry.
[0366] Modulation of either CD4 or the chemokine co-receptors are active strategies being tested in the clinic. A potential role for CXCR4 antagonists (e.g. AMD3100) in treatment of advanced stages of AIDS through inhibition of CXCR4 is anticipated, as X4 HIV-1 strains emerge late in this disease. To determine if the CXCR4#2 Nanobody is also capable of blocking the entry of CXCR4-using HIV1 strains, HIV-1 infection assays were performed with CXCR4 and CCRS specific HIV clones. The specificity of the inhibitory effects of the monovalent and bispecific CXC4-CD4 Nanobodies was tested on CXCR4-using (X4) HIV-1 clone NL4.3 infecting MT-4 cells, or freshly isolated PBMCs (CD4+/CXCR4+/CCR5+), and the CCRS-using (R5) HIV-1 strain BaL infecting freshly isolated PBMCs (CD4+/CXCR4+/CCRS+).
EXAMPLE 2.5.1
HIV-1 Infection Assays
[0367] The anti-HIV-1 potencies of the entire panel of bispecific CD4-CXCR4 polypeptides and the monovalent CXCR4#2 and CD4#8 Nanobodies were determined by measuring the cytopathic effect of distinct HIV-1 strains in MT-4 and U87 cell lines, or by quantification of the viral p24 antigen production in the culture supernatant of PBMCs.
[0368] Viral strains used were the X4 HIV-1 clone NL4.3, R5 HIV-1 strain BaL, or the R5/X4 HIV-1 HE strain. Infection was done in MT-4 cells or phytohemagglutin-stimulated PBMCs from different healthy donors. The CXCR4-using (X4) HIV-1 clone NL4.3 was obtained from the National Institutes of Health NIAID AIDS Reagent program (Bethesda, Md.), the CCR5-using (R5) HIV-1 strain BaL was obtained from the Medical Research Council AIDS reagent project (Herts, UK). The dual-tropic (R5/X4) HIV-1 HE strain was initially isolated from a patient at the University Hospital in Leuven. The MT-4 cells were seeded out in 96-well plate and the U87 cells in 24-well plates. Nanobodies were added at different concentrations together with HIV-1 and the plates were maintained at 37° C. in 10% CO.sub.2. Cytopathic effect induced by the virus was monitored by daily microscopic evaluation of the virus-infected cell cultures. At day 4-5 after infection, when strong cytopathic effect was observed in the positive control (i.e., untreated HIV-infected cells), the cell viability was assessed via the in situ reduction of the tetrazolium compound MTS, using the CellTiter 96® AQ.sub.ueous One Solution Cell Proliferation Assay (Promega, Madison, Wis.). The absorbance was measured spectrophotometrically at 490 nm with a 96-well plate reader (Molecular Devices, Sunnyvale, Calif.) and compared with four cell control replicates (cells without virus and drugs) and four virus control wells (virus-infected cells without drugs). The IC.sub.50, i.e., the drug concentration that inhibits HIV-induced cell death by 50%, was calculated for each polypeptide from the dose-response curve. The CC.sub.50 or 50% cytotoxic concentration of each of the polypeptides was determined from the reduction of viability of uninfected cells exposed to the agents, as measured by the MTS method described above.
[0369] Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density centrifugation (Lymphoprep; Nycomed Pharma, AS Diagnostics, Oslo, Norway) and stimulated with phytohemagglutin for 3 days. The activated cells were washed with PBS and viral infections were performed as described previously (Schols et al. J Exp Med 1997; 186:1383-1388). At 8-10 days after the start of the infection, viral p24 Ag was detected in the culture supernatant by an enzyme-linked immunosorbent assay (Perkin Elmer, Brussels, Belgium).
[0370] The HIV1 neutralisation results were depicted as IC.sub.50 values in Table 2.4. In MT-4 cells infected with the NL4.3 strain, the CXCR4#2 Nanobody specifically inhibited anti-X4 HIV1 entry via CXCR4, but not binding to CCR5. The CD4#8 Nanobody was effectively blocking both X4 HIV1 infection, with a similar IC50 value as the CXCR4 monovalent. In this example the CD4 Nanobody is not exclusively serving as anchor, but also contributes to the functional blockade. Bispecific CXCR4#2-CD4#8 polypeptides were extremely potent in inhibiting HIV-1 X4 virus replication, especially when evaluated in PHA-stimulated PBMCs. Potency increases for the best bispecific CXCR4-CD4 construct with the shortest linker were between 250-320 fold compared to monovalent CXCR4#2 Nanobody. Bispecific Nanobodies in the inverse orientation, with the reduced affinity towards CXCR4, were less active in this functional assay. Thus, the simultaneous binding to both CXCR4 and CD4 of the bispecific CXCR4-CD4 Nanobodies results in strongly enhanced potencies in the neutralization of CXCR4-using HIV1.
TABLE-US-00006 TABLE 2.4 Specificity for CXCR4-tropic HIV1 strain NL4.3 and the CCR5-tropic BaL. IC50 (nM) n = 3 MT-4 + U87 + PBMC + PBMC + Nanobody NL 4.3(X4) NL 4.3 (X4) NL 4.3(X4) BaL(R5) CD4#8 66.67 >1333 580 610 CXCR4#2 67.11 >6666 29.3 >1666 CD4#8-9GS-CXCR4#2 14.89 >3333 17.0 >666 CD4#8-25GS-CXCR4#2 9.22 >3333 8.67 383.33 CD4#8-35GS-CXCR4#2 11.67 >3333 23.7 35.9 CXCR4#2-9GS-CD4#8 0.20 0.53 0.03 CXCR4#2-25GS-CD4#8 0.21 2.67 0.12 CXCR4#2-35GS-CD4#8 0.24 2.67 0.37 2.46 AMD3100 4.75 10 1.91
Example 2.5.2
Specificity
[0371] The potency of the CXCR4 Nanobody is specific for HIV1 strains that depend on CXCR4 usage for entry. One potential disadvantage of blockade of only one HIV1 co-receptors is that it may trigger the re-emergence of the HIV subtype that is not originally targeted. In case of the CCR5-dependent HIV BaL virus, only the CD4 Nanobody in the bispecific construct contributes to the virus neutralization in PBMCs. Since CXCR4 is expressed on PBMCs, in these cells the CXCR4 Nanobody in the bispecific can serve as anchor to enhance the inhibition potency of the CD4 Nanobody. Indeed bispecific CXCR4-CD4 with the longest linker has an IC50 values of 2.5 nM for BaL, around 200-fold enhancements relative to monovalent CD4#8, and are more potent inhibitors of infection than constructs in the inverse orientation, where the CXCR4 binding affinity is impaired. For the CD4-CXCR4 bispecifics a longer linker appears to give better inhibition, suggesting that this favours the binding to the CXCR4 as anchor.
EXAMPLE 2.5.3
Entry-Inhibitor Resistant HIV-1 NL4.3
[0372] To substantiate the contribution of the CXCR4 Nanobody as anchor in the bispecific polypeptide, blockade of HIV infection was assessed for a panel of HIV1 mutant that was made resistant for the CXCR4 small molecule inhibitor AMD3100, the CXCR-4 ligand, or the control antibody 12G2. In addition, viral escape mutants were generated for blockade of each of the monovalent Nanobodies, by culturing of NL4.3 in presence of polypeptides at IC90 concentration over multiple passages. Resistant viral clones that were thus identified were used for testing the potencies of bispecific polypeptides compared to the monovalent polypeptides. IC50 values are presented in Table 2.5.
[0373] The IC50 values of the bispecific CXCR4-CD4 Nanobodies towards AMD3100 resistant virus are depicted in
TABLE-US-00007 TABLE 2.5 Anti-HIV activity profile of Nanobodies towards entry- inhibitor resistant HIV-1 NL4.3 variants in MT-4 cells. IC50 (M) MT-4 Nanobody NL4.3 wt CD4#8 res. CXCR4#2 res AMD-3100 res. CXCL-12res. 2G12 res. CD4#8 3.47E−08 >6.7E−06 >6.7E−06 2.27E−08 1.53E−07 2.33E−08 CXCR4#2 2.27E−08 8.73E−08 2.33E−06 >1.67E−06 2.20E−07 1.73E−08 CXCR4#2-35GS-CD4#8 1.87E−10 3.10E−10 1.40E−09 1.13E−09 4.33E−10 1.10E−10 CD4#8-35GS-CXCR4#2 6.00E−09 9.57E−08 >3.1E−07 1.40E−08 7.00E−08 3.00E−09 AMD3100 4.28E−09 1.85E−08 3.99E−07 4.04E−07 5.03E−08
TABLE-US-00008 TABLE 2.6 Anti-HIV activity profile of Nanobodies towards distinct HIV strains on MT-4 cells. IC50 (M) MT-4+ Nanobody HIV-1 NL4.3 HIV-1 HE HIV-2 ROD CD4#8 3.47E−08 1.00E−08 2.27E−08 CXCR4#2 2.27E−08 1.00E−08 8.67E−08 CXCR4#2-35GS-CD4#8 1.87E−10 9.06E−11 3.00E−10 CD4#8-35GS-CXCR4#2 6.00E−09 2.00E−09 8.75E−09 AMD3100 4.28E−09 3.90E−09 2.11E−08
EXAMPLE 3
Preferential Targeting of T Cell Subsets with CD4-IL12Rβ2 and CD4-IL23R bispecific Polypeptides
EXAMPLE 3.1
Characteristics of Monovalent Nanobodies Used for Formatting
[0374] To generate bispecific polypeptides with the capacity to preferential block specific T cell subsets, Nanobodies directed against different subset-specific interleukin receptors were combined with a Nanobody directed against the CD4 glycoprotein. On the functional arm the IL-12R132 was used as marker for the TH1 cell subset, and IL-23R as marker for T.sub.H17 cells. Both receptors belong to the same interleukin 12 receptor family and use the same co-receptor IL-12Rβ1 to form a functional heterodimer. For this reason also bispecific constructs of IL-12Rβ1 and CD4 were generated, as these are expected to target both T cell subsets and hence can serve as positive control. An anchoring Nanobody directed against the CD4 glycoprotein is used, to provide avidity and to prevent blockade of receptors on other immune cells, such as CD8+ T cells, B cells, natural killer cells and certain myeloid cells.
[0375] For this example, Nanobody 3F11 directed against the CD4 glycoprotein was used as common anchor. This Nanobody is specific for cell-expressed human CD4, and shows only low binding to recombinant CD4 protein, and was used in the generation of CXCR4-CD4 bispecifics (see Example 2). The Nanobodies specific for IL-23R, IL-12Rβ1 and IL-12Rβ2 were previously identified as ligand competing Nanobodies. To identify ligand-competing Nanobodies with sufficient low affinities for formatting, monovalent Nanobodies from families with multiple family members were characterised with respect to binding kinetics, ability to compete with ligand, and binding to cell-expressed receptors on primary cells.
EXAMPLE 3.1.1
SPR
[0376] The precise binding affinities of the purified Nanobodies were determined in a multi-cycle kinetic analysis using Surface Plasmon Resonance analysis (Biacore T100) on Fc-fusions of human IL12Rβ1, IL12Rβ2 and IL-23R extracellular domains (R&D Systems, #839-B1, #1959-B2, #1400-IR). Sensorchips CM5 were immobilized with anti-hIgG antibody (GE Healthcare, BR-1008-39), after which receptors were captured at 5 μg/ml protein and contact time of 120 seconds. Running buffer used was HBS-EP+ (GE Healthcare, BR-1006-69) at 25° C., with a flow-rate of 5 ml/min. For immobilization by amine coupling, EDC/NHS was used for activation and ethanolamine HCl for deactivation (Biacore, amine coupling kit). Nanobodies were evaluated at a concentration range between 1.37 nM and 3 μM. Nanobodies were allowed to associate for 2 min and to dissociate for 15 min at a flow rate of 45 ml/min. In between injections, the surfaces were regenerated with a 3 min pulse of 3M MgCl.sub.2 and 2 min stabilization period. Evaluation of the association/dissociation data was performed by fitting a 1:1 interaction model (Langmuir binding model) by Biacore TWO software v2.0.3. The off-rates and affinity constants are shown in Table 3.1.
EXAMPLE 3.1.2
Competition for IL-12 and IL-23 Binding in ELISA
[0377] The ability of monovalent Nanobodies to compete with binding of IL12 receptor-Fc proteins to IL-12 was assessed in a competition ELISA on coated human IL-12 (10 nM, Peprotech #200-12B) in a 384-well SpectraPlate HB microtiter plate (Perkin Elmer). Free binding sites were blocked with 1% casein in PBS. Serial dilutions of Nanobodies with a fixed concentration of either 2 nM IL12Rβ1-Fc or 3 nM IL12Rβ2-Fc were incubated for 1 hr. Concentration of competitors was based on dose-titration experiments, and final concentrations used were <EC.sub.50 values. Residual binding of IL12Rβ1-Fc or IL12Rβ2-Fc was detected using a HRP-conjugated goat anti-hIgG antibody ( 1/3000, Jackson ImmunoResearch, Cat#109-035-088) and a subsequent enzymatic reaction in the presence of the substrate esTMB (SDT reagents).
[0378] Similar assay set-ups were used for measuring the competition of IL-23R and IL12Rβ1 Nanobodies for binding to IL-23. A coating of human IL-23 (eBioscience 34-8239-82) at 20 nM was used for competition with 5 nM IL-23R-Fc, a coating of 3 nM was used for competition of 2 nM IL12Rβ1-Fc. The IC50 values are shown in Table 3.1. The difference in ligand competition ability between the family members for each of the IL12 receptor subunits correlates well with the difference in K.sub.D values measured.
EXAMPLE 3.1.3
Flow Cytometry
[0379] Dose-dependent binding of monovalent Nanobodies to their cell-expressed receptor in the context of the heterodimeric complex was determined by flow cytometry on activated human T cells from distinct healthy donors.
[0380] Human T cells were isolated using the Human T Cell Enrichment Cocktail (RosetteSep #15061) and pre-activated for four days with Dynabeads® Human T-Activator CD3/CD28 (Gibco—Life Technologies #11131D) and one day with recombinant human IL-2 (Life Technologies—Gibco #PHC0027) to induce TH1 differentiation. Routinely, T cell markers surface expression and activation state was checked by FACS using anti-CD3 PE (eBioscience #12-0037-73), anti-CD8-PE (BD Bioscience #555367), anti-CD45RO-PE (BD Bioscience #555493), anti-CD45RA-APC (BD Bioscience #550855) anti-CD25-PE (BD Bioscience #557138) and anti-CD69-PE (BD Bioscience #557050). IL12R surface expression was confirmed by FACS using IL12Rβ1 antibody (R&D MAB839), followed by goat anti-mouse PE (Jackson Immuno Research 115-115-164). The expression of IL23R was checked by polyclonal goat anti-IL-23R (R&D AF1400). CD4 surface expression was confirmed by FACS using APC-labelled anti-CD4 (BD Bioscience #345771). In
[0381] As the expression of IL23R was very low in the T cell pool, the binding of monovalent IL23R Nanobodies was assessed on cells that were differentiated towards the Th17 phenotype by the incubation of PBMCs in the presence of a cytokine cocktail and IL-23, recombinant IL-6 (eBioscience #34-8069-82), recombinant TGF-b1 (R&D #240-B), anti-human IL-4 antibody(BD#554481), recombinant IL-1b (BD#554602)) and recombinant Human IL-23 (R&D Systems #219-IL-005) with co-stimulation of plate coated OKT-3 (eBioscience #16-0037-85), PeliCluster CD28 (Sanquin #M1650). Following this procedure, low but detectable IL23R expression levels were obtained. Optimization in the Th17 differentiation protocol could further increase these expression levels.
[0382] Dose-dependent binding of monovalent Nanobodies was assessed by flow cytometry on the respective Th1 or Th17 enriched T cell populations. Serial dilutions of antibody or Nanobodies were allowed to associate for 30 minutes at 4° C. in FACS buffer (PBS supplemented with 10% FBS and 0.05% azide). Cells were washed by centrifugation and probed with anti-FLAG antibodies (Sigma F1804) for 30 minutes at 4° C., to detect bound Nanobody. Detection was done with Goat anti-Mouse IgG-PE (Jackson ImmunoResearch #115-116-071) for 30 minutes at 4° C. Cells were washed and incubated with TOPRO3 to stain for dead cells, which are then removed during the gating procedure. The cells were then analysed via a BD FACSArray.
[0383] Specific Nanobody binding curves are shown in
[0384] Specific binding of the IL-23R and IL12Rβ1 Nanobodies with the highest affinity was observed on the TH17-enriched population, although the fluorescence signals were very low (
[0385] The characteristics of the IL-23R, IL-12Rβ1 and IL-12Rβ2 Nanobodies selected for formatting into bispecific Nanobodies are presented in Table 3.1. We aimed to select Nanobodies with distinct off-rates belonging to the same family, i.e. with sequence conservation in their CDR3 regions, so that the epitope on the target was conserved and the effect of affinity could be addressed. Ideally Nanobodies with off-rates >10.sup.−4 s.sup.−1 were chosen, to maximise the avidity effect provided by the anchoring Nanobody. The sequences of the two selected IL12Rβ2 Nanobodies differ in three amino acids in CDR1 and CDR2 regions, and show a 3.5-fold difference in K.sub.D and ligand competition ability due to a difference in off-rate. The two selected IL12Rβ1 Nanobodies differ in six amino acids in the CDR1 and CDR3 regions, with a 6-7-fold difference in K.sub.D and ligand competition. For the IL23R Nanobodies it proved not feasible to identify two family members with a substantial difference in off-rate. Therefore for this receptor two ligand competing Nanobodies with different fast off-rates from distinct families, hence with potentially different epitopes, were selected. Although cell binding could not always be accurately measured for the Nanobodies with fast off-rates, (>1.E.sup.−02,) ligand competition assays demonstrated functional blocking with IC50 ranging between 10-16 nM for all selected monovalent Nanobodies.
EXAMPLE 3.2
Generation of Bispecific Nanobodies
[0386] Formatting of bi-specific CD4-IL-12Rβ2, CD4-IL-12Rβ1 and CD-IL-23R polypeptides was done by genetic fusion of Nanobodies linked with a long flexible (GGGGS)7 linker, with the building blocks in both orientations. For each combination, two functional blocking receptor-specific Nanobodies were combined with one anti-CD4 Nanobody, CD4#8 3F11 (
EXAMPLE 3.3
Binding Analysis of Bispecific Nanobodies
EXAMPLE 3.3.1
Effect of Formatting
[0387] To assess whether the orientation of the Nanobody after formatting affects the binding and functionality to the respective interleukin receptor, purified monovalent and bispecific Nanobodies were analysed for competition with either hIL-12Rβ1-Fc, hIL-12Rβ2-Fc or IL-23R-Fc fusions for ligand binding (see above). Dose-dependent inhibition of both monovalent Nanobodies and bispecifics was carried out to determine IC.sub.50 values for competition on plates coated with human IL-12. Similarly, a competition ELISA on plates coated with human IL-23 was performed to assess the functionality of bispecifics of the IL-23R and IL-12Rβ1 Nanobodies. The IC50 values are shown in Tables 3.2 and 3.3.
[0388] In case of CD4, orientation effects were assessed by flow cytometry, comparing binding of monovalent Nanobodies and bispecific polypeptides to MOLM-13 cells that express CD4 but lack IL12R and IL23R. The CD4 expression was confirmed by FACS using the anti-human CD4 APC (BD Bioscience, #53384).
EXAMPLE 3.3.2
Specificity
[0389] Dose-dependent binding of bispecific polypeptides was assessed on human T cells that were activated to increase expression levels of IL12R. Activated T cells showed relative moderate expression levels of the IL12R antigen, but very high CD4 expression, reflected in the high apparent affinity and high fluorescence signal of the anti-CD4 Nanobody. Simultaneous binding to the two target receptors is not apparent, as the binding curves of all bispecific Nanobodies overlap with those of the monovalent CD4 Nanobody, giving similar EC50 values (
[0390] The pool of activated T cells comprises both CD4+ T cells and CD8+ T cells. To confirm the specificity of the anti-CD4 Nanobody and to exclude binding to CD4-negative cells, binding was assessed to cytotoxic CD8+ T cells isolated from human PBMCs using the CD8+ T Cell Isolation Kit (Miltenyi Biotech, 130-096-495), resulting in 94% purity of CD8+ cells. Binding specificity experiments were carried out using Nanobodies at 250 nM. No binding was observed with the anti-CD4 Nanobody, while monovalent IL12Rb1#30 did bind to isolated CD8+ T cells (
[0391] To elucidate if CD4-IL12R bispecific polypeptides preferentially bind to the CD4+/IL12R+ T.sub.H1 cell subset within the pool of T cells, Nanobody binding was analysed to a pool of activated T cells gated for either CD8 (detected by Anti-hu CD8 PE-Cy7 conjugated monoclonal antibody (BD 557746) or CD4 (detected by Anti-hu CD4 alexa Fluor 488-conjugated polyclonal antibody (R&D FAB8165G) in a multi-colour FACS experiment. Nanobody binding to the CD8+/CD4− gated cells and to CD4+ gated cells was determined using anti-flag-APC (Prozyme PJ255) detection. In this experiment T.sub.H1 activated T cells from the same donor (D838) as shown in
EXAMPLE 3.4
Functional Characterization of Bispecific Polypeptides
EXAMPLE 3.4.1
Cell-Specific Blockade of IL-12 Function in Human T Cells
[0392] The ability of bispecific polypeptides to simultaneously engage both targets on the same cell was analysed in a IL-12 dependent functional assay, inhibition of IL-12 mediated IFN-γ release in activated human T cells. Since the functional blockade is only mediated via IL12R, avidity by the simultaneous binding of the CD4 Nanobody is expected to translate into increased potency of the bispecific in inhibition of cytokine release.
[0393] Isolated human T cells from buffycoats were activated for four days with Dynabeads® Human T-Activator CD3/CD28 (Gibco—Life Technologies #11131D) and one day with IL-2. To differentiate into Th1 subtype, T cells were cultured in presence of IL-12 with co-stimulation provided by plate coated CD3 at 0.5 μg/ml (eBioscience #16-0037-85) and anti-CD28 (1 μg/ml PeliCluster, Sanquin #M1650) in solution. Concentration of ligand used, 0.2 pM was based on dose—titration experiments, using concentration<EC50.As measure for IL-12 dependent signaling, release of the typical Th1 cytokine IFN-γ was measured after 72 h in the presence or absence of the respectively Nanobodies by ELISA.
[0394] Dose-dependent blockade of IL-12 mediated IFNγ release was assessed for the bispecific IL-12Rβ2-CD4 and IL-12Rβ1-CD4 polypeptides in both orientations, and the corresponding monovalent Nanobodies. The IL-23R-CD4 bispecific polypeptides served as negative controls. Representative graphs of the bispecific IL12Rβ1-CD4, IL12Rβ2-CD4 and IL23R-CD4 polypeptides are shown in
[0395] To verify if this selective functionality of the bispecific polypeptides on T.sub.H1 cells was preserved in PBMCs, where also other immune cells were present, the same assay was performed using activated healthy human PBMCs. T cells within the PBMC pool were differentiated towards the T.sub.H1 subtype using 0.1 pM IL-12. IFN-γ release in the presence or absence of the respectively Nanobodies was determined by ELISA after an incubation period of 6 days. A representative example of IL12 blockade of bispecific polypeptides in PBMCs is shown in
EXAMPLE 3.4.2
Cell-Specific Blockade of IL-23 Function
[0396] To verify if bispecific polypeptides targeting the functional IL23 receptor showed increased affinity and potency on cells that co-express CD4 and IL23R, the ability of Nanobodies to inhibit IL23-dependent release of the Th17 type cytokine IL17 was measured. In this assay set-up human PBMCs were cultured in the presence of soluble IL23 to allow differentiation of T cells towards the T.sub.h17 phenotype. Cells were seeded onto OKT-3 (eBioscience #16-0037-85) coated plates in the presence of recombinant human IL-23 (eBioscience #14-8239) and PeliCluster CD28 (Sanquin #M1650) in solution. Cytokine (IL17) release in the presence or absence of the respectively Nanobodies was determined by ELISA after an incubation period of 9 days.
[0397] Dose-dependent inhibition of the panel of bispecific IL23R-CD4 and IL12Rβ1-CD4 polypeptides was assessed in comparison to the respective monovalent Nanobodies, with in this case the IL12Rβ1-CD4 specific polypeptides serving as negative controls.
[0398] For the bispecific constructs of IL23R and CD4 there is also a difference in potency observed between monovalent Nanobodies and bispecific polypeptides (
[0399] Taken together, these results indicate that T.sub.H1-subset specific CD4-IL12Rβ2 and T.sub.H17-subset specific CD4-IL23R polypeptides show selective functional blockade in a T cell subset-specific manner, in assays with heterogeneous T cells as well as PBMCs. Furthermore, selective binding of the bispecific polypeptides to CD4+ T cell subsets was shown, whereas monovalent IL12Rβ2 Nanobodies showed only poor binding to CD4 and CD8 T cells.
[0400] With respect to affinities, even low affinity Nanobodies on the functional arm gave potency enhancements of 2-3 logs upon formatting with a high affinity anchoring CD4 Nanobody.
TABLE-US-00009 TABLE 3.1 Characteristics of monovalent IL-12Rb2, IL-12Rb2 and IL-23R-specific Nanobodies Inhibition of ligand binding Binding T cells Binding kinetics (SPR) (ELISA) (FACS) Nanobody ID k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) IC.sub.50 (M) EC.sub.50 (M) IL12Rb2#1: 135B08 2.1E+05 5.7E−04 2.7E−09 4.2E−09 1.5E−09 IL12Rb2#2: 135A07 2.7E+05 1.8E−03 6.9E−09 1.5E−08 1.8E−09 IL12Rb1#30: 148C09 4.5E+05 1.5E−03 3.3E−09 3.7E−09 (IL-12), 1.3E−09 1.5E−9 (IL-23) IL12Rb1#31: 148F09 7.2E+05 1.7E−02 2.3E−08 2.2E−08 (IL-12), No fit 1.0E−8 (IL-23) IL23R#19: 150D02 7.3E+05 8.1E−03 1.1E−08 4.7E−09 2E−08 IL23R#20: 150H07 3.0E+06 2.3E−01 7.8E−08 1.6E−08 No fit
TABLE-US-00010 TABLE 3.2 Inhibition of IL-12 function by panel of monovalent and bispecific IL12Rb1-CD4, IL12Rb2-CD4, and IL23R-CD4 Nanobodies. Inhibition of IL-12 comp IFNγ release T IFNγ release ELISA fold cells D839 fold PBMC D840 fold Nanobody ID Nb1 Nb2 IC50 (M) ctrl IC50 (M) ctrl IC50 (M) ctrl CD4#8 — — — IL23R#19 — BI#42 IL23R#19 CD4#8 BI#45 CD4#8 IL23R#19 — IL23R#20 — BI#43 IL23R#20 CD4#8 — — — BI#44 CD4#8 IL23R#20 — — — IL12Rb1#30 3.70E−09 1.40E−07 1.70E−08 BI#46 IL12Rb1#30 CD4#8 6.20E−09 0.6 2.9E−10 552 4.5E−10 38 BI#40 CD4#8 IL12Rb1#30 7.30E−09 0.5 2.8E−10 429 3.6E−10 47 IL12Rb1#31 2.20E−08 no fit 9.35E−08 BI#47 IL12Rb1#31 CD4#8 2.40E−08 0.9 4.1E−09 1.9E−09 41 BI#41 CD4#8 IL12Rb1#31 2.80E−08 0.8 1.7E−09 2.2E−09 50 IL12Rb2#1 4.20E−09 5.10E−08 1.35E−08 BI#37 IL12Rb2#1 CD4#8 4.80E−09 0.9 1.10E−10 400 1.1E−09 11 BI#39 CD4#8 IL12Rb2#1 1.00E−08 0.4 7.8E−10 74 1.9E−09 8 IL12Rb2#2 1.50E−08 6.70E−08 4.00E−08 BI#36 IL12Rb2#2 CD4#8 1.10E−08 1.4 3.1E−11 1129 1.6E−09 30 BI#38 CD4#8 IL12Rb2#2 2.50E−08 0.6 7.6E−10 130 2.1E−09 8
TABLE-US-00011 TABLE 3.3 Inhibition of IL-23 function by panel of monovalent and bispecific IL23R-CD4, IL12Rb1-CD4, and IL12Rb2-CD4 Nanobodies. Inhibition of IL-23 IL-23 comp IL-17 release Nanobody ELISA fold PBMC D840 fold ID Nb1 Nb2 IC50 (M) ctrl IC50 (M) ctrl CD4#8 — — IL23R#19 1.70E−09 1.00E−07 BI#42 IL23R#19 CD4#8 6.10E−09 0.3 5.89E−08 1.7 BI#45 CD4#8 IL23R#19 6.60E−09 0.3 2.42E−08 4.1 IL23R#20 1.60E−08 no fit BI#43 IL23R#20 CD4#8 1.10E−08 1.5 no fit BI#44 CD4#8 IL23R#20 1.50E−08 1.1 2.20E−08 IL12Rb1#30 1.50E−09 3.55E−08 BI#46 IL12Rb1#30 CD4#8 3.00E−09 0.5 1.6E−11 875 BI#40 CD4#8 IL12Rb1#30 3.60E−09 0.4 3.5E−11 1629 IL12Rb1#31 1.00E−08 2.10E−07 BI#47 IL12Rb1#31 CD4#8 1.30E−08 0.8 2.3E−10 565 BI#41 CD4#8 IL12Rb1#31 1.50E−08 0.7 1.7E−10 1706 IL12Rb2#1 BI#37 IL12Rb2#1 CD4#8 BI#39 CD4#8 IL12Rb2#1 IL12Rb2#2 BI#36 IL12Rb2#2 CD4#8 — — BI#38 CD4#8 IL12Rb2#2 — —
EXAMPLE 4
EGFR-CEA Bispecific Polypeptides
EXAMPLE 4.1
Characteristics of Monovalent Nanobodies Used for Formatting
[0401] Previous examples indicated that the cell-specific avidity of bispecific polypeptides can be measured by potency increase in functional assays, where bispecific polypeptides will block receptor function specifically on cells when they can simultaneously engage both targets in cis. To demonstrate the therapeutic window, functional cellular assays were done on cells that co-express the two targets (“double-positive cells”), and cells that only express the functional target (“single-positive cells”) representing normal cells.
[0402] Our previous examples also indicated that for the cell-specific blockade monovalent functional Nanobodies are needed with low affinities and potencies, to ensure that monospecific Nanobodies are not sufficiently potent on normal cells. To obtain selectivity very low affinities were needed, where the bispecific merely resembles the anchor, indicating there is a delicate trade-off between selectivity and sufficient functional potency. In the current example we further addressed the effect of affinity for Nanobodies on the functional arm, to determine if there is a threshold affinity for selective blockade. The tyrosine kinase receptor EGFR is used as model antigen on the functional arm, for which recombinant protein is available to allow the precise determination of the affinities and kinetic parameters by SPR.
[0403] The second target, carcinoembryonic antigen (CEA, also known as CEACAMS), is a well-known tumour specific antigen expressed on many tumour types. CEA is a glycosylphosphatidylinisotol (GPI)-anchored cell surface glycoprotein that plays a role in cellular adhesion. It is an established tumour-associated marker for gastrointestinal tract cancers and also found in breast and lung cancers. Co-expression of EGFR and CEA has been reported for gastric and colorectal cancers, in primary tumours and in peritoneal metastasis, with in most cases higher membrane expression of CEA than EGFR (Ito et al. 2013, Tiernan et al. 2013). This makes CEA a useful target to serve as anchor for combining with EGFR for functional blockade in a tumour-selective manner.
[0404] Ligand-blocking Nanobodies against EGFR were previously generated in-house and well described by Roovers et al. (2011). Nanobody 7D12 binds to the ligand binding site on domain III of the extracellular domain of EGFR, overlapping with the epitope of cetuximab. The reported affinity of [.sup.125I] radiolabelled 7D12 was 10.4 and 25.7 nM for HER14 and A431 cells, respectively. Its family member 7C12 differs in 5 amino acid residues.
[0405] To assess the effect of affinity while ensuring that the epitope on EGFR was preserved, a panel of EGFR 7D12 and 7C12 variants with reduced affinities was generated for use in formatting. Based on the co-crystal structure of Nanobody 7D12 with the EGFR ectodomain (Schmitz et al., 2013), amino acids in the receptor interface in 7D12 were substituted with residues that were expected to reduce the off-rates in a step-wise manner (Table 4.1).
[0406] On the anchoring arm, a CEACAM5-specific Nanobody designated NbCEA5 was used with a reported high affinity of K.sub.D 0.3 nM by Cortez-Ramiras et al. (2004). A variant of this Nanobody has been described with a 30-fold reduction in its affinity due to introduction of the CDR regions into a human scaffold (Vaneycken et al., 2011). Both Nanobodies as well as additional CEA variants were generated with a number of amino acid substitutions, to reduce the affinity but safe-guard a sufficiently high Nanobody expression (Table 4.2).
[0407] The panel of monovalent EGFR 7D12 variants and NbCEA5 variants with decreased affinities was characterised with respect to binding kinetics, and binding to cell-expressed receptors.
EXAMPLE 4.1.1
SPR
[0408] To determine the precise binding affinities of the purified EGFR variants, a multi-cycle kinetic analysis was performed using Surface Plasmon Resonance analysis (Biacore T100) on directly immobilized hEGFR extracellular domain (Sino Biological, #10001-H08H). Around 1000RU of hEGFR was immobilized on a CM5 sensor chip. Running buffer used was HBS-EP+ (GE Healthcare, BR-1006-69) at 25° C., with a flow-rate of 5 μl/min. For immobilization by amine coupling, EDC/NHS was used for activation and ethanolamine HCl for deactivation (Biacore, amine coupling kit). Nanobodies were evaluated at a concentration range between 1.37 nM and 3 μM. Nanobodies were allowed to associate for 2 min and to dissociate for 15 min at a flow rate of 45 μl/min. In between injections, the surfaces were regenerated with a 5 sec pulse of 50 mM NaOH and 1 min stabilization period. Evaluation of the association/dissociation data was performed by fitting a 1:1 interaction model (Langmuir binding model) by Biacore T100 software v2.0.3. Interactions which did not meet the acceptance criteria for the 1:1 interaction model, were fitted using the heterogeneous ligand fit model. The affinity constant K.sub.D was calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d, and are shown in Table 4.1. The introduction of defined amino acid substitutions clearly reduced the off-rate of the EGFR Nanobody, while on-rates were similar.
[0409] The binding affinities of the purified CEA Nanobodies were obtained using similar experimental conditions on directly immobilized hCEACAM-5 (R&D Systems, #4128-CM) up to 1000RU on a CM5 sensor chip. In between injections, the surfaces were regenerated with a 5 sec pulse of 10 mM Glycine-HCl pH1.5 and 1 min stabilization period. Evaluation of the association/dissociation data was performed by fitting a 1:1 interaction model (Langmuir binding model) by Biacore T100 software v2.0.3. The affinity constant K.sub.D was calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d and are shown in Table 4.2. The observed affinity of the NbCEA5 Nanobody (designated as CEA#1) and humanised variant (CEA#2) was in line with the reported value.
EXAMPLE 4.1.2
Binding to Recombinant EGFR and CEACAM5 Proteins in ELISA
[0410] All purified Nanobodies were shown to bind to the recombinant EGFR ectodomain and to recombinant CEACAM5 protein in a dose dependent manner in binding ELISA. In short, 0.25 μg/ml of human EGFR ECD (Sino Biological, Cat#10001-H08H) or 0.125 μg/ml recombinant human CEACAM5 (R&D Systems, Cat #4128-CM) were coated directly on 384-well SpectraPlate-HB microtiter plates (Perkin Elmer). Free binding sites were blocked with 1% casein in PBS. Serial dilutions of purified Nanobodies were allowed to bind the antigen for 1 hour. Nanobody binding was detected using HRP conjugated mouse-anti-FLAG M2 antibody (Sigma, Cat#A8592) and a subsequent enzymatic reaction in the presence of the substrate esTMB (SDT reagents, Cat#esTMB). Binding specificity was determined based on OD values compared to irrelevant Nanobody controls. The EC50 values are shown in Tables 4.1 and 4.2.
EXAMPLE 4.1.3
FACS Binding
[0411] The colon carcinoma cell lines LoVo and HT-29 co-express EGFR and CEA with different relative expression levels (
[0412] The binding characteristics of the monovalent EGFR 7D12 variants and CEA variants are presented in Tables 4.1 and 4.2, respectively. For the generation of EGFR-CEA bispecific Nanobodies, four EGFR variants were selected with differences in off-rates resulting in gradual decreased K.sub.D values (ranging between 120-860 nM). The gap in off-rate between the highest and lowest affinity EGFR variant was 8-fold. When measured in ELISA, the difference was enlarged to ˜80-fold, due to dissociation of the Nanobodies with the fast off-rates during the washing. Compared to the highest affinity variant EGFR#1, variant EGFR#11 has two amino acid substitutions, whereas EGFR#33 and EGFR#32 have three amino acid differences. For the anchoring arm, besides the original CEA Nanobody (CEA#1), also CEA variant#5 was selected for use in formatting, with four amino acid substitutions, as this Nanobody had the largest difference in off-rate compared to the original Nanobody.
TABLE-US-00012 TABLE 4.1 Binding characteristics of monovalent EGFR Nanobodies used for formatting ELISA EGFErbB-1-PY Nanobody hEGFR ECD hEGFR on Her-14 ID Description ka (1/Ms) kd (1/s) KD (M) EC.sub.50 (M) IC50 (M) EGFR#1 7C12 (A1E, A14T, T98A, Q108L) 2.1E+05 2.4E−02 1.2E−07 7.1E−10 7.9E−08 EGFR#10 7C12 (Q108L) 4.1E+05* 1.7E−02* 4.1E−08 EGFR#11 7C12 (A1E, Q108L) 2.2E+05 3.7E−02 1.7E−07 3.3E−09 7.1E−08 EGFR#12 7C12(E100fS, Q108L) 1.9E+05 8.0E−02 4.3E−07 EGFR#13 7C12(Y102A, Q108L) 3.4E+05* 8.6E−01* 2.5E−06* EGFR#16 7C12(R27S, Q108L) 2.4E+05 2.1E−02 9.0E−08 EGFR#33 7C12(A1E, R27S, Q108L) 2.2E+05 5.4E−02 2.4E−07 4.5E−09 2.4E−07 EGFR#32 7C12(A1E, E100fS, Q108L) 2.7E+05 2.3E−01 8.6E−07 5.8E−08 1.4E−06 *Indicative values
TABLE-US-00013 TABLE 4.2 Characteristics of monovalent CEACAM5 Nanobodies used for formatting ELISA FACS Nanobody hCEACAM5 CEACAM5 LoVo ID Description ka (1/Ms) kd (1/s) KD (M) EC50 (M) EC.sub.50 (M) CEA#1 NbCEA5 9.9E+05 5.1E−04 5.1E−10 2.6E−11 1.0E−9 CEA#2 NbCEA5(S11L, A14P, K43Q, E44G, 1.3E+06 2.5E−03 1.9E−09 R45L, G47A, T73N, A74S, V78L, P84A, D85E, D89V) CEA#5 NbCEA5(K43Q, G47A, T73N, V78L) 1.1E+06 3.3E−03 3.1E−09 1.1E−10 2.4E−9
EXAMPLE 4.2
Generation of Bispecific Polypeptides
[0413] Formatting of bispecific EGFR-CEA polypeptides was accomplished by genetic fusion of Nanobodies linked with a flexible 35GS linker, with both building blocks in both orientations. Four different EGFR variants with distinct off-rates were combined with two distinct CEA Nanobodies with K.sub.D values of 0.5 and 3 nM, respectively (
EXAMPLE 4.3
Binding Analysis of Bispecific EGFR-CEA Nanobodies
EXAMPLE 4.3.1
Effect of Formatting
[0414] To verify if the formatting affected the ability of each of the building blocks to bind their respective target, binding of the purified bispecific Nanobodies was assessed by means of binding ELISA on recombinant EGFR ectodomain or CEACAM5, as described above. The EC50 values of all monospecific Nanobodies and bispecific polypeptides comprising EGFR-CEA are shown in Table 4.3.
[0415] For all EGFR-CEA bispecifics, the CEA Nanobody retained similar binding as the respective monovalent Nanobody. In contrast, the EGFR Nanobodies were sensitive to the position within the bispecific construct, and only in the N-terminal position the interaction with EGFR is preserved (
EXAMPLE 4.3.2
Binding Specificity
[0416] Binding specificities of the monospecific and bispecific EGFR-CEA Nanobody constructs were analysed by flow cytometry on EGFR+/CEA− HER14 and HeLa cells, and double-positive LoVo and HT-29 cells, respectively. EC50 values are presented in Table 4.3. Results for LoVo cells are shown in
[0417] Bispecific polypeptides efficiently bound to cells in a dose-dependent manner. In line with the ELISA data, the bispecific polypeptides with the EGFR#1 Nanobody in C-terminal position lost substantial binding affinity on both HER14 and LoVo cells. When comparing the monospecific EGFR Nanobodies, the differences in off-rates between the distinct EGFR variants are less pronounced on cell-expressed EGFR, especially when EGFR expression levels are not so high, such as on LoVo and HeLa cells (
[0418] On LoVo cells, bispecific polypeptides in the EGFR-CEA orientation showed increased fluorescence levels and a slight shift in EC50 values compared to the respective EGFR control Nanobodies (
TABLE-US-00014 TABLE 4.3 Binding analysis of monospecific and bispecific EGFR-CEA bispecific Nanobodies. EGFR CEA Her-14 HeLa LoVo ELISA ELISA EGFR++/CEA− EGFR+/CEA− EGFR+/CEA+ ID Description EC50 (M) EC50 (M) EC50 (M) EC50 (M) EC50 (M) BI#52 EGFR#1- ctrl 1.30E−09 — 8.6E−09 3.9E−09 4.3E−09 BI#26 EGFR#1-CEA#1 6.70E−10 4.90E−11 7.5E−09 1.9E−09 1.5E−09 BI#27 EGFR#1-CEA#5 3.50E−10 1.20E−10 2.0E−09 2.1E−09 3.4E−09 BI#28 CEA#1-EGFR#1 2.30E−08 5.60E−11 3.4E−08 2.4E−07 1.1E−09 BI#29 CEA#5- EGFR#1 1.30E−08 1.00E−10 3.6E−08 1.3E−07 3.0E−09 BI#49 EGFR#11- ctrl 1.70E−09 — 9.1E−09 2.9E−09 5.0E−09 BI#22 EGFR#11-CEA#1 9.35E−10 4.70E−11 7.1E−09 2.5E−09 1.5E−09 BI#24 EGFR#11-CEA#5 8.30E−10 1.40E−10 4.3E−09 3.6E−09 3.8E−09 BI#53 EGFR#33- ctrl 4.00E−09 — 1.7E−08 3.5E−09 4.5E−09 BI#34 EGFR#33-CEA#1 1.30E−09 5.30E−11 1.1E−08 2.7E−08 2.1E−09 BI#35 EGFR#33-CEA#5 6.10E−10 8.90E−11 3.6E−09 4.0E−09 3.7E−09 BI#50 EGFR#32-ctrl 1.60E−08 — 2.0E−08 5.7E−08 5.6E−08 BI#23 EGFR#32-CEA#1 1.10E−08 4.60E−11 1.4E−08 7.8E−08 1.8E−09 BI#25 EGFR#32-CEA#5 6.00E−09 1.20E−10 1.5E−08 1.1E−07 4.9E−09 BI#48 CEA#1-ctrl — 2.60E−11 — — 9.2E−10 BI#51 CEA#5-ctrl — 1.10E−10 — — 2.6E−09
EXAMPLE 4.4
Inhibition of EGFR Function by Bispecific EGFR-CEA Polypeptides
[0419] To verify if bispecific polypeptides could enhance the potency of the EGFR Nanobodies by simultaneously engagement of EGFR and CEA on the cell surface, the panel of bispecific EGFR-CEA polypeptides and corresponding monospecific Nanobodies was analysed in a functional EGFR assay.
[0420] Dose-dependent inhibition of EGFR phosphorylation was assessed on HER14 cells expressing only EGFR, and EGFR+/CEA+ LoVo cells. Since the functional phosphorylation is only mediated via EGFR, avidity by the simultaneous binding of the CEA Nanobody is expected to translate into increased inhibition of EGFR phosphorylation in a cell-specific manner.
[0421] Briefly, LoVo cells were seeded in duplicate into 96-well culture plates at 2×10.sup.4 cells per well in F12-K medium supplemented with 10% FCS. HER14 cells were seeded in duplicate into 0.1% gelatin coated 96-well culture plates and grown in DMEM culture medium containing 10% FBS/BS for 24 h. The next day, cells were serum-starved in medium supplemented with 0.1% FCS for 24 hrs and then incubated with Nanobodies followed by stimulation for 10 minutes with 0.5 nM of recombinant human EGF (R&D Systems, cat#236-EG) for HER14 and 1 nM for LoVo cells. EGF concentrations were based on the EC50 obtained in LoVo (EC50=5.9 ng/ml) and HER14 cells (EC50=3.5 ng/ml). In each plate anti-EGFR mAb cetuximab (Erbitux Merck-Serono) and irrelevant control Nanobodies were included as reference. Monolayers were rinsed twice with ice-cold dPBS, and subsequently lysed in ice cold RIPA buffer substituted with 1 mM PMSF. EGF-dependent receptor activation in cell lysates was measured using a Phospho(Tyr1173)/Total EGFR Whole Cell Lysate Kit (Meso Scale Discovery—K15104D). Plates were loaded with 30 μl of lysate, incubated 1 h at RT with shaking and processed according to the manufacturer's protocol. Plates were read on the Sector Imager 2400 (Meso Scale Discovery). The percentage of phospho-protein over total protein was calculated using the formula:
(2×p-protein)/(p-protein+total protein)×100.
[0422] Representative graphs are shown in
[0423] On EGFR+/CEA+ LoVo cells, about 5 fold difference in potency between monospecific and bispecific EGFR-CEA polypeptides was observed for constructs with EGFR#1 and EGFR#33 combined with the CEA#1 Nanobody as anchor. Constructs with the lowest affinity EGFR#32 variant could not block EGFR function, and the additional presence of CEA Nanobody could not enhance its potency.
[0424] Taken together, these results show that potency enhancements were obtained with bispecific polypeptides for the EGFR and CEACAM5 target combination, exclusively on cells that co-express both receptors. The relative small potency increase of EGFR-CEA bispecific polypeptides observed on LoVo cells in the phosphorylation assay may be related to a suboptimal ratio between CEA and EGFR expression on this cells, but it is also possible that the potency effects will be larger in assays that measure functional responses of EGF, such as proliferation and survival. Besides the effect on receptor phosphorylation at one timepoint, as assessed in the current assay, the Nanobody could have differential effects on the receptor inactivation and degradation kinetics, which are not be assessed in a signal transduction assay. It is also possible that the selected Nanobodies for this example had sterical limitations with respect to the epitope on the target, which may restrict simultaneous engagement of both targets on the cell surface.
[0425] A gain in potency was observed for the current combination tested but bispecific EGFR-CEA polypeptides directed towards other epitopes may show larger in cell-specific potency enhancements
TABLE-US-00015 TABLE 4.4 Inhibition of EGF-mediated EGFR phosphorylation by EGFR-CEA bispecific Nanobodies compared to monospecific control Nanobodies. HER-14 (n = 2) LoVo (n = 2-3) EGFR+/CEA− EGFR+/CEA+ fold fold ID Description IC50 (M) increase* IC50 (M) increase BI#52 EGFR#1- ctrl 6.55E−08 7.53E−08 BI#26 EGFR#1-CEA#1 5.80E−08 1.1 1.50E−08 5.0 BI#27 EGFR#1-CEA#5 3.65E−08 1.8 2.50E−08 3.0 BI#28 CEA#1-EGFR#1 1.75E−06 4.83E−06 BI#29 CEA#5-EGFR#1 1.95E−06 3.10E−06 BI#49 EGFR#11- ctrl 5.25E−08 1.50E−07 BI#22 EGFR#11-CEA#1 6.25E−08 0.8 4.30E−08 3.5 BI#24 EGFR#11-CEA#5 5.00E−08 1.1 4.15E−08 3.6 BI#53 EGFR#33- ctrl 1.50E−07 4.63E−07 BI#34 EGFR#33-CEA#1 6.05E−08 2.5 5.98E−08 7.7 BI#35 EGFR#33-CEA#5 1.23E−07 1.2 8.90E−08 5.2 BI#50 EGFR#32-ctrl 6.90E−07 2.85E−06 BI#23 EGFR#32-CEA#1 1.50E−06 0.7 8.85E−07 3.2 BI#25 EGFR#32-CEA#5 8.10E−07 1.3 8.08E−07 3.5 BI#48 CEA#1-ctrl — — erbitux 1.25E−09 4.4E−10 *IC50 ratio relative to respective monospecific EGFR Nanobodies on same cell line.
TABLE-US-00016 TABLE 1 CXCR4 building blocks SEQ ID NO Amino acid sequence 14D09 1 EVQLVESGGGLVQAGGSLRLSCVAS GISSSKRNMGWYRQAPGKQRESVAT ISSGGNKDYTDAVKDRFTISRDTTK NTVYLQMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTQVTVSS 14A09 2 EVQLVESGGGLVQAGGSLRLSCVAS GISSSIRNSGWYRQAPGKQRESVAT ISSGGNKDYTDAVKDRFTISRDTTK NTVYLQMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTQVTVSS 281F12 3 EVQLVESGGGLVQAGDSLRLSCAAS (Q108L) GRAFSRYAMGWFRQAPGKEREFVAA IGWGPSKTNYADSVKGRFTISRDNA KNTVYLQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGTLVTVS S 14A02 4 EVQLVESGGGLVQAGGSLRLSCVAS GISSSIRNMGWYRQAPGKQRESVAT ISSGGNKDYTDAVKDRFTXSRDTTK NTVYLQMSSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTQVTVSS 14E02 5 EVQLVESGGGLVQAGGSLRLSCVAS GISSSIRNMGWYRQAPGKQRESVAT ISSGGNKDYTDAVKDRFTISRDTTK NTVYLQMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTQVTVSS 14D09 6 EVQLVESGGGLVQAGGSLRLSCVAS (Q108L) GISSSKRNMGWYRQAPGKQRESVAT ISSGGNKDYTDAVKDRFTISRDTTK NTVYLQMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTLVTVSS 281F12 4CXCR281F1 7 EVQLVESGGGLVQAGDSLRLSCAAS (TAG) 2-FLAG3- GRAFSRYAMGWFRQAPGKEREFVAA HIS6 IGWGPSKTNYADSVKGRFTISRDNA KNTVYLQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGTLVTVS SAAADYKDHDGDYKDHDIDYKDDDD KGAAHHHHHH 14D09 4CXCR014D0 8 EVQLVESGGGLVQAGGSLRLSCVAS (TAG) 9-FLAG3- GISSSKRNMGWYRQAPGKQRESVAT HIS6 ISSGGNKDYTDAVKDRFTISRDTTK NTVYLQMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTLVTVSSAAA DYKDHDGDYKDHDIDYKDDDDKGAA HHHHHH
TABLE-US-00017 TABLE 2 CD123 building blocks SEQ ID NO Amino acid sequence 55B04 9 EVQLVESGGGLVQPGGSLRLSCAAS GINFRFNSMGWWRRRAPGKEREWVA AITSGDITNYRDSVRGRFTISRDNV KNTVYLQMNTLKLEDTAVYYCNTFP PIADYWGLGTQVTVSS 51D09 10 EVQLVESGGGLVQPGGSLRLSCAAS GSIFSGNTMGWYRQAPGKQRELVAA ISSGGSTDYADSVKGRFTISRDNSK NTVYLQMNSLRPEDTAVYYCNAAIL LYRLYGYEEGDYWGLGTLVTVSS 55C05 11 EVQLVESGGGLVPAGDSLRLSCVAS GRSLNTYTMGWFRQAPGKECEFVAA INWNGVYRDYADSAKGRFTASRDNA MNTVFLQMNSLKPEDTAVYFCATAT QGWDRHTEPSDFGSWGLGTQVTVSS 50F07 12 EVQLVESGGGLVQPGGSLRLSCTGS GSTFSINAMGWYRQAPGKQRELVAA ITSGGRTNYADSVKGRFTISRDNSK NTVYLQMNSLRPEDTAVYYCNARIS AGTAFWLWSDYEYWGLGTLVTVSS 55F03 13 EVQLVESGGGLVQAGGPLRLSCAAS GRTFSSYVMGWFRQAPGKEREFVAA IYWSNGKTQYTDSVKGRFTISGDNA KNTVYLQMNSLNPEDTAVYYCVADK DETGFRTLPIAYDYWGLGTQVTVSS 55A01 14 EVQLVESGGGSVQAGGSLRLSCTTS GRALNMYVMGWFRQAPGNEREFVAA TSSSGGSTSYPDSVKGRFTISRDNA KNTVYLQMNSLKPEDTAAYRCAASP YVSTPTMNILEEYRYWGLGTQVTVS S 57A07 15 EVQLVESGGGLVQAGGSLRLSCAAS GSIFSGNVMGWYRRQAPGKEREWVA AIASGGSIYYRDSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYYCNSHP PTLPYWGLGTQVTVSS
TABLE-US-00018 TABLE 3 Characteristics of monovalent IL-3Ra Nanobodies SPR - IL-3Ra FACS binding mAb 7G3 Nanobody KD MOLM-13 THP-1 Hek-IL-R3a competition ID Germ-line ka (1/Ms) kd (1/s) [M] EC50 (M) EC50 (M) EC50 (M) IC50 (M) CD123#1 57A07 VHH2 1.0E+06 8.1E−04 7.83E−10 6.6E−10 1.3E−9 2.4E−10 1.20E−09 CD123#2 55A01 VHH3 8.4E+04 1.4E−03 1.71E−08 8.2E−9 1.1E−8 1.10E−09 4.00E−08 55B04 VHH2 5.04E+05 7.94E−03 1.58E−08 5.12E−08 5.50E−08 51D09 VHH2 3.78E+04 5.16E−04 1.36E−08 1.53E−08 55C05 VHH3 1.26E+05 7.41E−03 5.90E−08 3.12E−08 1.90E−07 50F07 VHH2 1.02E+05 7.58E−03 7.42E−08 1.46E−08 2.30E−07 55F03 VHH3 4.25E+04 4.87E−03 1.15E−07 1.13E−07
TABLE-US-00019 TABLE 4 Characteristics of monovalent CXCR4 Nanobodies Ligand competition Chemotaxis CXCR4 Binding Nanobody Biotin-SDF-1 [.sup.125I]- SDF-1 # Jurkat Caki-CXCR4 Jurkat CXCR4-VLP Description ID Fam IC50 (nM) Ki (nM) IC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM) CXCR4#2 281F12 3 26.9 68 nd 7.8 nd CXCR4#1 14D09 57 18.4 11 9.9 11 7.28 14A02 57 4.1 0.95 4.0 1.15 0.73 14E02 57 13.5 2.6 nd 0.78 238D4 17 2.1 5.4 7.6 3.4 nd nd # Determined with [.sup.125I]- SDF-1 on membrane extracts of Hek-CXCR4 cells.
TABLE-US-00020 TABLE 5 Summary of selected Nanobodies Medium affinity: High affinity: EC50/kD 1 < x <= 10 nM Medium/low potency Nanobody K.sub.D <= 1 nM Ligand inhibition Ligand inhibition function Target EC.sub.50 <= 1 nM IC50 1 < x < 10 nM IC50 >= 10 nM Functional CXCR4 CXCR4#1: 14D09 CXCR4#2: 281F12 Anchor CD123 CD123#1: 57A07 CD123#2: 55A01 Anchor CD4 CD4#8: 3F11 Functional IL12Rβ1 IL12Rβ1#30: 148C09 IL12Rβ1#31: 148F09 Functional IL12Rβ2 IL12Rβ2#1: 135B08 IL12Rβ2#2: 135A07 Functional IL-23R IL23R#19: 150D02 IL23R#20: 150H07 Functional EGFR EGFR#1/11/33/32: 7D12 variants Anchor CEACAM5 CEA#1: NbCEA5 CEA#5: NbCEA5 variant
TABLE-US-00021 TABLE 9 High affinity: Nanobody K.sub.D <= 1 nM Medium affinity: function Target EC.sub.50 <= 1 nM EC50 1 < x <= 10 nM Anchor CD123 CD123#1: 57A07 CD123#2: 55A01 Anchor CD4 CD4#8: 3F11 Anchor CEACAM5 CEA#1: NbCEA5 CEA#5: NbCEA5 variant
TABLE-US-00022 TABLE 6 Summary of bispecific constructs 57A07 - 14D09 55A01 - 14D09 57A07 - 281F12 55A01 - 281F12 14D09 - 57A07 14D09 - 55A01 281F12 - 57A07 281F12 - 55A01
TABLE-US-00023 TABLE 7 bispecific constructs (all with c-myc HIS6 tag) SEQ ID NO Amino acid sequence 57A07- A0110057A0 16 EVQLVESGGGLVQAGGSLRL 14D09 7-35GS- SCAASGSIFSGNVMGWYRRQ 4CXCR014D APGKEREWVAAIASGGSIYY 09(Q108L) RDSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCNSHP PTLPYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGG GLVQAGGSLRLSCVASGISS SKRNMGWYRQAPGKQRESVA TISSGGNKDYTDAVKDRFTI SRDTTKNTVYLQMNSLKPED TAVYYCKIEAGTGWATRRGY TYWGQGTLVTVSSAAAEQKL ISEED1NGAAHHHHHH 57A07- A0110057A0 17 EVQLVESGGGLVQAGGSLRL 281F12 7-35GS- SCAASGSIFSGNVMGWYRRQ 4CXCR281F1 APGKEREWVAAIASGGSIYY 2(Q108L) RDSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCNSHP PTLPYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGG GLVQAGDSLRLSCAASGRAF SRYAMGWFRQAPGKEREFVA AIGWGPSKTNYADSVKGRFT ISRDNAKNTVYLQMNTLKPE DTAVYSCAAKFVNTDSTWSR SEMYTYWGQGTLVTVSSAAA EQKLISEEDLNGAAHHHHHH 14D09- 4CXCR014D 18 EVQLVESGGGLVQAGGSLRL 57A07 09(Q108L)- SCVASGISSSKRNMGWYRQA 35GS- PGKQRESVATISSGGNKDYT A0110057A0 DAVKDRFTISRDTTKNGVYL 7- QMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTLVTV SSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQ LVESGGGLVQAGGSLRLSCA ASGSIFSGNVMGWYRRQAPG KEREWVAAIASGGSIYYRDS VKGRFTISRDNAKNTVYLQM NSLKPEDTAVYYCNSHPPTL PYWGQGTLVTVSSAAAEQKL ISEEDLNGAAHHHHHH 281F12- 4CXCR281F1 19 EVQLVESGGGLVQAGDSLRL 57A07 2(Q108L)- SCAASGRAFSRYAMGWFRQA 35GS- PGKEREFVAAIGWGPSKTNY A0110057A0 ADSVKGRFTISRDNAKNTVY 7- LQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGT LVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGG SEVQLVESGGGLVQAGGSLR LSCAASGSIFSGNVMGWYRR QAPGKEREWVAAIASGGSIY YRDSVKGRFTISRDNAKNTV YLQMNSLKPEDTAVYYCNSH PPTLPYWGQGTLVTVSSAAA EQKLISEEDLNGAAHHHHHH 55A01- A0110055A0 20 EVQLVESGGGSVQAGGSLRL 14D09 1-35GS- SCTTSGRALNMYVMGWFRQA 4CXCR014D PGNEREFVAATSSSGGSTSY 09(Q108L) PDSVKGRFTISRDNAKNTVY LQMNSLKPEDTAAYRCAASP YVSTPTMNILEEYRYWGQGT LVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGG SEVQLVESGGGLVQAGGSLR ESCVASGISSSKRNMGWYRQ APGKQRESVATISSGGNKDY TDAVKDRFTISRDTTKNTVY LQMNSLKPEDTAVYYCKIEA GTGWATRRGYTYWGQGTLVT VSSAAAEQKLISEEDLNGAA HHHHHH 55A01- A0110055A0 21 EVQLVESGGGSVQAGGSLRL 281F12 1-35GS- SCTTSGRALNMYVMGWFRQA 4CXCR281F1 PGNEREFVAATSSSGGSTSY 2(Q108L) PDSVKGRFTISRDNAKNTVY LQMNSLKPEDTAAYRCAASP YVSTPTMNILEEYRYWGQGT LVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGG SEVQLVESGGGLVQAGDSLR LSCAASGRAFSRYAMGWFRQ APGKEREFVAAIGWGPSKTN YADSVKGRFTISRDNAKNTV YLQMNTLKPEDTAVYSCAAK FVNTDSTWSRSEMYTYWGQG TLVTVSSAAAEQKLISEEDL NGAAHHHHHH 14D09- 4CXCR014D 22 EVQLVESGGGLVQAGGSLRL 55A01 09(Q108L)- SCVASGISSSKRNMGWYRQA 35GS- PGKQRESVATISSGGNKDYT A0110055A0 DAVKDRFTISRDTTKNTVYL 1 QMNSLKPEDTAVYYCKIEAG TGWATRRGYTYWGQGTLVTV SSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQ LVESGGGSVQAGGSLRLSCT TSGRALNMYVMGWFRQAPGN EREFVAATSSSGGSTSYPDS VKGRFTISRDNAKNTVYLQM NSLKPEDTAAYRCAASPYVS TPTMNILEEYRYWGQGTLVT VSSAAAEQKLISEEDLNGAA HHHHHH 281F12- 4CXCR281F1 23 EVQLVESGGGLVQAGDSLRL 55A01 2(Q108L)- SCAASGRAFSRYAMGWFRQA 35GS- PGKEREFVAAIGWGPSKTNY A0110055A0 ADSVKGRFTISRDNAKNTVY 1 LQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGT LVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGG SEVQLVESGGGSVQAGGSLR LSCTTSGRALNMYVMGWFRQ APGNEREFVAATSSSGGSTS YPDSVKGRFTISRDNAKNTV YLQMNSLKPEDTAAYRCAAS PYVSTPTMNILEEYRYWGQG TLVTVSSAAAEQKLISEEDL NGAAHHHHHH
TABLE-US-00024 TABLE 8 Potencies of monovalent and bispecific CXCR4-IL3Ra Nanobodies ® to inhibit CXCL-12 induced chemotaxis. Jurkat E6-1 MOLM-13 Abbreviation N-terminal C-terminal IC50 95% LCI 95% UCI Fold inc. IC50 95% LCI 95% UCI Fold inc. CXCR4#1 14D09 — 1.04E−08 7.08E−09 1.56E−08 — 8.62E−09 5.33E−09 1.61E−08 — CXCR4#1- 14D09 57A07 1.50E−08 1.00E−08 2.35E−08 0.69 3.60E−09 2.50E−09 5.35E−09 2.39 CD123#1 CXCR4#1- 14D09 55A01 1.20E−08 8.20E−09 1.70E−08 0.87 3.60E−09 2.25E−09 5.70E−09 2.39 CD123#2 CD123#1- 57A07 014D09 2.10E−07 1.60E−07 2.80E−07 0.05 — — — — CXCR4#1 CD123#2- 55A01 014D09 8.50E−08 3.90E−08 1.90E−07 0.12 2.90E−07 1.70E−07 4.90E−07 0.03 CXCR4#1 CXCR4#2 281F12 — 9.68E−08 7.08E−08 1.33E−07 — 8.60E−08 5.11E−08 1.51E−07 — CXCR4#2- 281F12 57A07 3.80E−08 2.40E−08 6.00E−08 2.55 6.83E−09 4.30E−09 1.20E−08 12.58 CD123# 1 CXCR4#2-I 281F12 55A01 8.60E−08 4.50E−08 1.73E−07 1.13 7.85E−09 5.55E−09 1.30E−08 10.95 CD123#2 CD123#1- 57A07 281F12 — — — — — — — — CXCR4#2 CD123#2- 55A01 281F12 — — — — — — — — CXCR4#2 Legend: IC50—average of the respective IC50 in 2-3 independent experiments LCI—Lower limit of 95% confidence interval (average from 2-3 independent experiments) UCI—Upper limit of 95% confidence interval (average from 2-3 independent experiments) Fold inc—fold increase of the bispecific construct compared to the respective anti-CXCR4 building block
TABLE-US-00025 TABLE 10 CXCR4-CD4 sequences SEQ Nanobody Code used ID ID in text NO Amino acid sequence 281F12 CXCR4#2 24 EVQLVESGGGLVQAGDSLRL SCAASGRAFSRYAMGWFRQA PGKEREFVAAIGWGPSKTNY ADSVKGRFTISRDNAKNTVY LQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGT QVTVSS 03F11 CD4#8 25 EVQLVESGGGSVQPGGSLTL SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTQ VTVSS 03F11-9GS- CD4#8-9GS- 26 EVQLVESGGGSVQPGGSLTL 281F12 CXCR4#2 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTQ VTVSSGGGGSGGGGSEVQLV ESGGGLVQAGDSLRLSCAAS GRAFSRYAMGWFRQAPGKER EFVAAIGWGPSKTNYADSVK GRFTISRDNAKNTVYLQMNT LKPEDTAVYSCAAKFVNTDS TWSRSEMYTYWGQGTQVTVS S 03F11-25GS- CD4#8-25GS- 27 EVQLVESGGGSVQPGGSLTL 281F12 CXCR4#2 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTQ VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGG LVQAGDSLRLSCAASGRAFS RYAMGWFRQAPGKEREFVAA IGWGPSKTNYADSVKGRFTI SRDNAKNTVYLQMNTLKPED TAVYSCAAKFVNTDSTWSRS EMYTYWGQGTQVTVSS 03F11-35GS- CD4#8-35GS- 28 EVQLVESGGGSVQPGGSLTL 281F12 CXCR4#2 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTQ VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQAGDSLRL SCAASGRAFSRYAMGWFRQA PGKEREFVAAIGWGPSKTNY ADSVKGRFTISRDNAKNTVY LQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGT QVTVSS 281F12-9GS- CXCR482-9GS- 29 EVQLVESGGGLVQAGDSLRL 03F11 CD4#8 SCAASGRAFSRYAMGWFRQA PGKEREFVAAIGWGPSKTNY ADSVKGRFTISRDNAKNTVY LQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGT QVTVSSGGGSGGGGSEVQLV ESGGGSVQPGGSLTLSCGTS GRTFNVMGWFRQAPGKEREF VAAVRWSSTGIYYTQYADSV KSRFTISRDNAKNTVYLEMN SLKPEDTAVYYCAADTYNSN PARWDGYDFRGQGTQVTVSS 281F12- CXCR4#2- 30 EVQLVESGGGLVQAGDSLRL 25GS-03F11 25GS- SCAASGRAFSRYAMGWFRQA CD4#8 P GKEREFVAAIGWGPSKTNYA DSVKGRFTISRDNAKNTVYL QMNTLKPEDTAVYSCAAKFV NTDSTWSRSEMYTYWGQGTQ VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGG SVQPGGSLTLSCGTSGRTFN VMGWFRQAPGKEREFVAAVR WSSTGIYYTQYADSVKSRFT ISRDNAKNTVYLEMNSLKPE DTAVYYCAADTYNSNPARWD GYDFRGQGTQVTVSS 281F12- CXCR4#2-35GS- 31 EVQLVESGGGLVQAGDSLRL 35GS-03F11 CD4#8 SCAASGRAFSRYAMGWFRQA PGKEREFVAAIGWGPSKTNY ADSVKGRFTISRDNAKNTVY LQMNTLKPEDTAVYSCAAKF VNTDSTWSRSEMYTYWGQGT QVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGG SEVQLVESGGGSVQPGGSLT LSCGTSGRTFNVMGWFRQAP GKEREFVAAVRWSSTGIYYT QYADSVKSRFTISRDNAKNT VYLEMNSLKPEDTAVYYCAA DTYNSNPARWDGYDFRGQGT QVTVSS A011000025 4CXCR281F12(L108Q)-35GS-4CD003F11 (L108Q)-FLAG3-HIS6 A011000026 4CD003F11(L108Q)-35GS-4CXCR281F12 (L108Q)-FLAG3-HIS6
TABLE-US-00026 TABLE 11 EGFR-CEA sequences SEQ Code ID Nb ID used: NO Amino acid sequence NbCEA5 CEA#1 32 EVQLVESGGGSVQAGGSLRL SCAASGDTYGSYWMGWFRQA PGKEREGVAAINRGGGYTVY ADSVKGRFTISRDTAKNTVY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSS T023200002 CEA#2 33 EVQLVESGGGLVQPGGSLRL SCAASGDTYGSYWMGWFRQA PGQGLEAVAAINRGGGYTVY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAASG VLGGLHEDWFNYWGQGTLVT VSS T023200003 CEA#3 34 EVQLVESGGGSVQAGGSLRL SCAASGDTYGSYWMGWFRQA PGKEREGVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSS T023200004 CEA#4 35 EVQLVESGGGSVQAGGSLRL SCAASGDTYGSYWMGWFRQA PGQEREGVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSS T023200005 CEA#5 36 EVQLVESGGGSVQAGGSLRL SCAASGDTYGSYWMGWFRQA PGQEREAVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSS T023200006 CEA#6 37 EVQLVESGGGSVQAGGSLRL SCAASGDTYGSYWMGWFRQA PGQELEAVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSS T023200007 CEA#7 38 EVQLVESGGGSVQAGGSLRL SCAASGDTYGSYWMGWFRQA PGQGLEAVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSS 7D12 EGFR#1 39 EVQLVESGGGSVQTGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSAWYGTLYEYDYWGQGTLV TVSS T023200010 EGFR#10 40 AVQLVESGGGSVQAGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSS T023200011 EGFR#11 41 EVQLVESGGGSVQAGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSS T023200012 EGFR#12 42 AVQLVESGGGSVQAGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSS T023200013 EGFR#13 43 AVQLVESGGGSVQAGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDAWGQGTLV TVSS T023200032 EGFR#32 44 EVQLVESGGGSVQAGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSS T023200033 EGFR#33 45 EVQLVESGGGSVQAGGSLRL TCAASGSTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSS T023200022 EGFR#11- 46 EVQLVESGGGSVQAGGSLRL CEA#1 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200023 EGFR#32- 47 EVQLVESGGGSVQAGGSLRL CEA#1 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200024 EGFR#11- 48 EVQLVESGGGSVQAGGSLRL CEA#5 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200025 EGFR#32- 49 EVQLVESGGGSVQAGGSLRL CEA#5 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200026 EGFR#1- 50 EVQLVESGGGSVQTGGSLRL CEA#1 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSAWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200027 EGFR#1- 51 EVQLVESGGGSVQTGGSLRL CEA#5 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSAWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SSDTAIYYCAAAAGSTWYGT LYEYDYWGQGTLVTVSS T023200022 EGFR#11- 46 EVQLVESGGGSVQAGGSLRL CEA#1 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200023 EGFR#32- 47 EVQLVESGGGSVQAGGSLRL CEA#1 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200024 EGFR#11- 48 EVQLVESGGGSVQAGGSLRL CEA#5 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200025 EGFR#32- 49 EVQLVESGGGSVQAGGSLRL CEA#5 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200026 EGFR#1- 50 EVQLVESGGGSVQTGGSLRL CEA#1 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSAWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200027 EGFR#1- 51 EVQLVESGGGSVQTGGSLRL CEA#5 TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSAWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200034 EGFR#33- 52 EVQLVESGGGSVQAGGSLRL CEA#1 TCAASGSTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GKEREGVAAINRGGGYTVYA DSVKGRFTISRDTAKNTVYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200035 EGFR#33- 53 EVQLVESGGGSVQAGGSLRL CEA#5 TCAASGSTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQAGGSLRLS CAASGDTYGSYWMGWFRQAP GQEREAVAAINRGGGYTVYA DSVKGRFTISRDNAKNTLYL QMNSLRPDDTADYYCAASGV LGGLHEDWFNYWGQGTLVTV SS T023200028 CEA#1- 54 EVQLVESGGGSVQAGGSLRL EGFR#1 SCAASGDTYGSYWMGWFRQA PGKEREGVAAINRGGGYTVY ADSVKGRFTISRDTAKNTVY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSEV QLVESGGGSVQTGGSLRLTC AASGRTSRSYGMGWFRQAPG KEREFVSGISWRGDSTGYAD SVKGRFTISRDNAKNTVDLQ MNSLKPEDTAIYYCAAAAGS AWYGTLYEYDYWGQGTLVTV SS T023200029 CEA#5- 55 EVQLVESGGGSVQAGGSLRL EGFR#1 SCAASGDTYGSYWMGWFRQA PGQEREAVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSEV QLVESGGGSVQTGGSLRLTC AASGRTSRSYGMGWFRQAPG KEREFVSGISWRGDSTGYAD SVKGRFTISRDNAKNTVDLQ MNSLKPEDTAIYYCAAAAGS AWYGTLYEYDYWGQGTLVTV SS T023200048 CEA#1- 56 EVQLVESGGGSVQAGGSLRL ctrl SCAASGDTYGSYWMGWFRQA PGKEREGVAAINRGGGYTVY ADSVKGRFTISRDTAKNTVY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSDV QLQASGGGSVQAGGSLRLSC AASGYTIGPYCMGWFRQAPG KEREGVAAINMGGGITYYAD SVKGRFTISQDNAKNTVYLL MNSLEPEDTAIYYCAADSTI YASYYECGHGLSTGGYGYDS WGQGTQVTVSS T023200051 CEA#5- 57 EVQLVESGGGSVQAGGSLRL ctrl SCAASGDTYGSYWMGWFRQA PGQEREAVAAINRGGGYTVY ADSVKGRFTISRDNAKNTLY LQMNSLRPDDTADYYCAASG VLGGLHEDWFNYWGQGTLVT VSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSDV QLQASGGGSVQAGGSLRLSC AASGYTIGPYCMGWFRQAPG KEREGVAAINMGGGITYYAD SVKGRFTISQDNAKNTVYLL MNSLEPEDTAIYYCAADSTI YASYYECGHGLSTGGYGYDS WGQGTQVTVSS T023200049 EGFR#11- 58 EVQLVESGGGSVQAGGSLRL ctrl TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSD VQLQASGGGSVQAGGSLRLS CAASGYTIGPYCMGWFRQAP GKEREGVAAINMGGGITYYA DSVKGRFTISQDNAKNTVYL LMNSLEPEDTAIYYCAADST IYASYYECGHGLSTGGYGYD SWGQGTQVTVSS T023200050 EGFR#32- 59 EVQLVESGGGSVQAGGSLRL ctrl TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYSYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSD VQLQASGGGSVQAGGSLRLS CAASGYTIGPYCMGWFRQAP GKEREGVAAINMGGGITYYA DSVKGRFTISQDNAKNTVYL LMNSLEPEDTAIYYCAADST IYASYYECGHGLSTGGYGYD SWGQGTQVTVSS T023200052 EGFR#l-ctrl 60 EVQLVESGGGSVQTGGSLRL TCAASGRTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSAWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSD VQLQASGGGSVQAGGSLRLS CAASGYTIGPYCMGWFRQAP GKEREGVAAINMGGGITYYA DSVKGRFTISQDNAKNTVYL LMNSLEPEDTAIYYCAADST IYASYYECGHGLSTGGYGYD SWGQGTQVTVSS T023200053 EGFR#33- 61 EVQLVESGGGSVQAGGSLRL ctrl TCAASGSTSRSYGMGWFRQA PGKEREFVSGISWRGDSTGY ADSVKGRFTISRDNAKNTVD LQMNSLKPEDTAIYYCAAAA GSTWYGTLYEYDYWGQGTLV TVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSD VQLQASGGGSVQAGGSLRLS CAASGYTIGPYCMGWFRQAP GKEREGVAAINMGGGITYYA DSVKGRFTISQDNAKNTVYL LMNSLEPEDTAIYYCAADST IYASYYECGHGLSTGGYGYD SWGQGTQVTVSS c-terminal CEA#1 CEA#5 EGFR#1 control n-terminal EGFR#1 BI#26 BI#27 BI#52 EGFR#11 BI#22 BI#24 BI#49 EGFR#32 BI#23 BI#25 BI#50 EGFR#33 BI#34 BI#35 BI#53 CEA#1 BI#28 BI#48 CEA#5 BI#29 BI#51
TABLE-US-00027 TABLE 12 CD4-IL12R CD4-IL23R sequences SEQ Code used ID Nb ID in text NO Amino acid Sequence 03F11 CD4#8 62 EVQLVESGGGSVQPGGSLTL SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSS LG150C02 IL23R#18 63 EVQLVESGGGLVQSGGSLRL SCAASEGTFTIYPLGWFRQA PGKDRKFVAALPWSAGIPQY SDSVKGRFTISRDNAKNTVY LQMNNLKPEDTAVYYCAAKG RDDSYQPWNYWGQGTLVTVS S LG150D02 IL23R#19 64 EVQLVESGGGLVQPGGSLTL SCVASGRTFSTDVMGWFRQA PGKEREFVAAHRTSGISTVY AASVKGRFTISRDNAKNTVY LGMKSLKPEDTAVYVCAAGS DASGGYDYWGQGTLVTVSS LG150H07 IL23R#20 65 EVQLVESGGGLVQAGGSLRL SCAASGRTFSSYAMGWFRQA PGKDREFVAAISWIGESTYY ADSVKGRFTISRDNAKNTVY LRMNSLKPEDTAVYYCAADL YYTAYVAAADEYDYWGQGTL VTVSS LG148C09 IL12Rb1#30 66 EVQLVESGGGLVQTGGSLRL SCAASGRTPRLVAMGWFRQT PGKEREFVGEIILSKGFTYY ADSVKGRFTISRVNAKNTIT MYLQMNSLKSEDTAVYYCAG RQNWSGSPARTNEYEYWGQG TLVTVSS LG148F09 IL12Rb1#31 67 EVQLVESGGGLVQTGGSLRL SCAASGRTPSIIAMGWFRQT PGKEREFVGEIILSKGFTYY ADSVKGRFTISRANAKNTIT MYLQMNSLKSEDTAVYYCAA RQNWSGNPTRTNEYEYWGQG TLVTVSS LG135B08 IL12Rb2#1 68 EVQLVESGGRLVQAGDSLRL SCAASGRTFISYRMGWFRQA PGKEREFVAALRWSSSNIDY TYYADSVKGRFSISGDYAKN TVYLQMNSLKAEDTAVYYCA ASTRWGVMESDTEYTSWGQG TLVTVSS LG135A07 IL12Rb2#2 69 EVQLVESGGRLVQAGDSLRL SCAASGRTFTSYRMGWFRQA PGKEREFVSALRWSSGNIDY TYYADSVKGRFSISGDYAKN TVYLQMNSLKAEDTAVYYCA ASTRWGVMESDTEYTSWGQG TLVTVSS T023200036 IL12Rb2#1- 70 EVQLVESGGRLVQAGDSLRL CD4#8 SCAASGRTFISYRMGWFRQA PGKEREFVAALRWSSSNIDY TYYADSVKGRFSISGDYAKN TVYLQMNSLKAEDTAVYYCA ASTRWGVMESDTEYTSWGQG TLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGSVQPGGSL TLSCGTSGRTFNVMGWFRQA PGKEREFVAAVRWSSTGIYY TQYADSVKSRFTISRDNAKN TVYLEMNSLKPEDTAVYYCA ADTYNSNPARWDGYDFRGQG TLVTVSS T023200037 IL12Rb2#2- 71 EVQLVESGGRLVQAGDSLRL CD4#8 SCAASGRTFTSYRMGWFRQA PGKEREFVSALRWSSGNIDY TYYADSVKGRFSISGDYAKN TVYLQMNSLKAEDTAVYYCA ASTRWGVMESDTEYTSWGQG TLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGSVQPGGSL TLSCGTSGRTFNVMGWFRQA PGKEREFVAAVRWSSTGIYY TQYADSVKSRFTISRDNAKN TVYLEMNSLKPEDTAVYYCA ADTYNSNPARWDGYDFRGQG TLVTVSS T023200038 CD4#8- 72 EVQLVESGGGSVQPGGSLTL IL12Rb2#1 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGRLVQAGDSLRL SCAASGRTFISYRMGWFRQA PGKEREFVAALRWSSSNIDY TYYADSVKGRFSISGDYAKN TVYLQMNSLKAEDTAVYYCA ASTRWGVMESDTEYTSWGQG TLVTVSS T023200039 CD4#8- 73 EVQLVESGGGSVQPGGSLTL IL12Rb2#2 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGRLVQAGDSLRL SCAASGRTFTSYRMGWFRQA PGKEREFVSALRWSSGNIDY TYYADSVKGRFSISGDYAKN TVYLQMNSLKAEDTAVYYCA ASTRWGVMESDTEYTSWGQG TLVTVSS T023200040 CD4#8- 74 EVQLVESGGGSVQPGGSLTL IL12Rb1#30 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQTGGSLRL SCAASGRTPRLVAMGWFRQT PGKEREFVGEIILSKGFTYY ADSVKGRFTISRVNAKNTIT MYLQMNSLKSEDTAVYYCAG RQNWSGSPARTNEYEYWGQG TLVTVSS T023200041 CD4#8- 75 EVQLVESGGGSVQPGGSLTL IL12Rb1#31 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQTGGSLRL SCAASGRTPSIIAMGWFRQT PGKEREFVGEIILSKGFTYY ADSVKGRFTISRANAKNTIT MYLQMNSLKSEDTAVYYCAA RQNWSGNPTRTNEYEYWGQG TLVTVSS T023200042 IL23R#19- 76 EVQLVESGGGLVQPGGSLTL CD4#8 SCVASGRTFSTDVMGWFRQA PGKEREFVAAHRTSGISTVY AASVKGRFTISRDNAKNTVY LGMKSLKPEDTAVYVCAAGS DASGGYDYWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSEVQLVE SGGGSVQPGGSLTLSCGTSG RTFNVMGWFRQAPGKEREFV AAVRWSSTGIYYTQYADSVK SRFTISRDNAKNTVYLEMNS LKPEDTAVYYCAADTYNSNP ARWDGYDFRGQGTLVTVSS T023200043 IL23R#20- 77 EVQLVESGGGLVQAGGSLRL CD4#8 SCAASGRTFSSYAMGWFRQA PGKDREFVAAISWIGESTYY ADSVKGRFTISRDNAKNTVY LRMNSLKPEDTAVYYCAADL YYTAYVAAADEYDYWGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGGSVQPGGSLTL SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSS T023200044 CD4#8- 78 EVQLVESGGGSVQPGGSLTL IL23R#20 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQAGGSLRL SCAASGRTFSSYAMGWFRQA PGKDREFVAAISWIGESTYY ADSVKGRFTISRDNAKNTVY LRMNSLKPEDTAVYYCAADL YYTAYVAAADEYDYWGQGTL VTVSS T023200045 CD4#8- 79 EVQLVESGGGSVQPGGSLTL IL23R#19 SCGTSGRTFNVMGWFRQAPG KEREFVAAVRWSSTGIYYTQ YADSVKSRFTISRDNAKNTV YLEMNSLKPEDTAVYYCAAD TYNSNPARWDGYDFRGQGTL VTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLTL SCVASGRTFSTDVMGWFRQA PGKEREFVAAHRTSGISTVY AASVKGRFTISRDNAKNTVY LGMKSLKPEDTAVYVCAAGS DASGGYDYWGQGTLVTVSS T023200046 IL12Rb1#30- 80 EVQLVESGGGLVQTGGSLRL CD4#8 SCAASGRTPRLVAMGWFRQT PGKEREFVGEIILSKGFTYY ADSVKGRFTISRVNAKNTIT MYLQMNSLKSEDTAVYYCAG RQNWSGSPARTNEYEYWGQG TLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGSVQPGGSL TLSCGTSGRTFNVMGWFRQA PGKEREFVAAVRWSSTGIYY TQYADSVKSRFTISRDNAKN TVYLEMNSLKPEDTAVYYCA ADTYNSNPARWDGYDFRGQG TLVTVSS T023200047 IL12Rb1#31- 81 EVQLVESGGGLVQTGGSLRL CD4#8 SCAASGRTPSIIAMGWFRQT PGKEREFVGEIILSKGFTYY ADSVKGRFTISRANAKNTIT MYLQMNSLKSEDTAVYYCAA RQNWSGNPTRTNEYEYWGQG TLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGSVQPGGSL TLSCGTSGRTFNVMGWFRQA PGKEREFVAAVRWSSTGIYY TQYADSVKSRFTISRDNAKN TVYLEMNSLKPEDTAVYYCA ADTYNSNPARWDGYDFRGQG TLVTVSS c-terminal CD4#8 IL12Rb2#1 IL12Rb2#2 n-terminal CD4#8 BI#38 BI#39 IL12Rb2#1 BI#36 IL12Rb2#2 BI#37 c-terminal CD4#8 IL12Rb1#30 IL12Rb1#31 n-terminal CD4#8 BI#40 BI#41 IL12Rb1#30 BI#46 IL12Rb1#31 BI#47 c-terminal CD4#8 IL23R#19 IL23R#20 n-terminal CD4#8 BI#45 BI#44 IL23R#19 BI#42 IL23R#20 BI#43
TABLE-US-00028 TABLE 13 CXCR4-CD123 Nanobody ID Code used in text: A011000003 4CXCR281F12(Q108L)-35GS-A0110055A01-CMYC-HIS6 CXCR4#2-CD123#5 A011000004 4CXCR281F12(Q108L)-35GS-A0110057A07-CMYC-HIS6 CXCR4#2-CD123#7 A011000007 4CXCR014D09(Q108L)-35GS-A0110055A01-CMYC-HIS6 CXCR4#1-CD123#5 A011000008 A0110055A01-35GS-4CXCR014D09(Q108L)-CMYC-HIS6 CD123#5-CXCR4#1 A011000010 A0110057A07-35GS-4CXCR281F12(Q108L)-CMYC-HIS6 CD123#7-CXCR4#2 A011000011 A0110057A07-35GS-4CXCR014D09(Q108L)-CMYC-HIS6 CD123#7-CXCR4#1 A011000015 A0110055A01-35GS-4CXCR281F12(Q108L)-CMYC-HIS6 CD123#5-CXCR4#2 A011000016 4CXCR014D09(Q108L)-35GS-A0110057A07-CMYC-HIS6 CXCR4#1-CD123#7 A011000017 4CXCR281F12-35GS-A0110055A01-FLAG3-HIS6 CXCR4#2-CD123#5 A011000018 4CXCR281F12-35GS-A0110057A07-FLAG3-HIS6 CXCR4#2-CD123#7 A011000019 4CXCR014D09-35GS-A0110055A01-FLAG3-HIS6 CXCR4#1-CD123#5 A011000020 4CXCR014D09-35GS-A0110057A07-FLAG3-HIS6 CXCR4#1-CD123#7 A011000021 A0110057A07-35GS-4CXCR281F12-FLAG3-HIS6 CD123#7-CXCR4#2 A011000022 A0110055A01-35GS-4CXCR281F12-FLAG3-HIS6 CD123#5-CXCR4#1 A011000023 A0110057A07-35GS-4CXCR014D09-FLAG3-HIS6 CD123#7-CXCR4#1 A011000024 A0110055A01-35GS-4CXCR014D09-FLAG3-HIS6 CD123#5-CXCR4#2 A011000025 4CXCR281F12(L108Q)-35GS-4CD003F11(L108Q)-FLAG3-HIS6 CXCR#2-CD4#8 A011000026 4CD003F11(L108Q)-35GS-4CXCR281F12(L108Q)-FLAG3-HIS6 CD4#2-CXCR4#2 A011000027 4CXCR281F12-Flag3-His6 CXCR4#2 A011000028 4CXCR014D09-Flag3-His6 CXCR4#1 c-terminal n-terminal CXCR4#2 CXCR4#1 CD123#7 CD123#5 CXCR4#2 BI#4/18 BI#3/17 CXCR4#1 BI#16/20 BI#7/19 CD123#7 BI#10/21 BI#11/23 CD123#5 BI#15/24 BI#8/22
TABLE-US-00029 TABLE B-4 Albumin binder sequences of the invention SEQ ID Name NO: Amino acid sequence Alb11 82 EVQLVESGGGLVQPGNSLRL SCAASGFTFSSFGMSWVRQA PGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLY LQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSS Alb8 83 EVQLVESGGGLVQPGNSLRL SCAASGFTFSSFGMSWVRQA PGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLY LQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSAAAEQ KLISEEDLNGAAHHHHHH
TABLE-US-00030 TABLE B-5 Linker sequences of the invention Name SEQ of ID Amino acid linker NO: sequences 5GS 84 GGGGS 6GS 85 SGGSGGS 9GS 86 GGGGSGGGS 10GS 87 GGGGSGGGGS 15GS 88 GGGGSGGGGSGGGGS 18GS 89 GGGGSGGGGSGGGGGGGS 20G5 90 GGGGSGGGGSGGGGSGGGGS 25G5 91 GGGGSGGGGSGGGGSGGGGSGGGGS 30G5 92 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35G5 93 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS