Anti-CD30 monoclonal antibodies and chimeric antigen receptors

Abstract

Antibodies, fragments thereof, and chimeric proteins comprising same are presented that have specific binding activity against CD30. Advantageously, contemplated molecules can be used in pharmaceutical compositions for immune therapy, particularly in individuals diagnosed with hematopoietic malignancies, including Hodgkin lymphoma, CD30-positive B cell lymphomas, CD30-positive T cell lymphomas, CD30-positive NK cell lymphomas.

Claims

1. A method of treatment for cancer in an individual in need thereof, the method comprising administering to the individual a pharmaceutical composition comprising an isolated antibody or fragment thereof, wherein the antibody or fragment thereof binds to CD30, the antibody or fragment thereof comprising: a variable heavy chain (V.sub.H) domain and a variable light chain (V.sub.L) domain; wherein the V.sub.H domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, and SEQ ID NO:5; and wherein the V.sub.L domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

2. The method of claim 1, wherein the antibody is an IgG.sub.1 antibody or an scFv.

3. The method of claim 1, further comprising administering a therapeutic agent.

4. The method of claim 3, wherein the therapeutic agent is a cancer vaccine, a chemotherapeutic drug, a radionuclide, or an immune stimulant.

5. The method of claim 4, wherein the immune stimulant is a cytokine, a cytokine analog, a chemokine, or a checkpoint inhibitor.

6. The method of claim 1, wherein the composition is administered by subcutaneous, subdermal, or intravenous injection.

7. The method of claim 1, wherein the composition is administered by intratumoral injection.

8. The method of claim 1, wherein administering the pharmaceutical composition results in an increased systemic concentration of at least one cytokine selected from the group consisting of IL-2, IL-12, IFN-?, IL-12, IL-23, IL-113, IL-6, and TGF-?.

9. The method of claim 1, further comprising administering a natural killer (NK) cell.

10. The method of claim 9, wherein the NK cell is an NK-92 cell.

11. The method of claim 1, wherein the cancer expresses CD30.

12. The method of claim 1, wherein the cancer is a hematological cancer.

13. The method of claim 1, wherein the cancer is a T cell lymphoma.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0019] FIG. 1 is a graph depicting exemplary FACS results for anti-CD30 antibody binding on cells expressing CD30 and control cells (HL-60) using a commercially available anti-CD30 antibody and an IgG1 antibody according to the inventive subject matter.

[0020] FIG. 2 is a graph depicting exemplary titration results for anti-CD30 antibody binding on cells expressing CD30 and control cells (HL-60) using a commercially available anti-CD30 antibody and an IgG1 antibody according to the inventive subject matter.

[0021] FIG. 3 is a graph depicting exemplary results for CAR specific target cell lysis of CD30 expressing target cells using haNK cells expressing exemplary anti-CD30 CAR constructs according to the inventive subject matter and a mock CAR.

[0022] FIG. 4 is a graph depicting exemplary results for CAR specific target cell lysis of control target cells not expressing CD30 using haNK cells expressing exemplary anti-CD30 CAR constructs according to the inventive subject matter and a mock CAR.

[0023] FIG. 5 is a graph depicting exemplary results for CD30 expression in the target cells of FIGS. 3 and 4.

[0024] FIG. 6 is a graph depicting exemplary results for anti-CD30 CAR and mock CAR expression in the haNK cells of FIGS. 3 and 4

[0025] FIG. 7 is a graph depicting exemplary results for CAR specific target cell lysis of CD30 expressing target cells (low, high, pool) using haNK cells expressing another exemplary anti-CD30 CAR construct according to the inventive subject matter and a mock CAR.

[0026] FIG. 8 is a graph depicting exemplary results for CD30 expression in the target cells of FIG. 7.

DETAILED DESCRIPTION

[0027] The inventors have discovered various anti-CD30 antibodies that have high affinity and specificity with respect to binding to CD30. In particularly preferred aspects, contemplated antibodies are human IgG.sub.1 antibodies that have the V.sub.H and V.sub.L domains as shown below. However, it should be appreciated that the sequences presented herein can vary to at least some degree and may therefore have one or more amino acid substitutions, insertions, and/or deletions as is discussed in more detail below. Most typically, but not necessarily, V.sub.H and V.sub.L domains, or heavy and light chains with the same preceding numeral (e.g., 52-2) will be present in a CD30 binding construct. However, other CD30 binding constructs may only have the V.sub.H or V.sub.L domain, or a V.sub.H and a V.sub.L domain with non-identical preceding numeral. Moreover, CD30 binding constructs may include those that have at least some of the CDRs (e.g., at least those from V.sub.H domain) as listed below.

TABLE-US-00001 52-2V.sub.Hdomainaminoacidsequence: (SEQIDNO:1) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMHWVRQAPGKGLEWVSA ISWSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR FMPFIPNTLGFDVWGQGTLVTVSS 52-2V.sub.Ldomainaminoacidsequence: (SEQIDNO:2) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYV ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDADVPLTFGQ GTKVEIK 52-5V.sub.Hdomainaminoacidsequence: (SEQIDNO:3) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMHWVRQAPGKGLEWVSA ISWSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR FMPFIPNTLGFDVWGQGTLVTVSS 52-5V.sub.Ldomainaminoacidsequence: (SEQIDNO:4) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDADVPLTFGQ GTKVEIK 52-38V.sub.Hdomainaminoacidsequence: (SEQIDNO:5) MEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVS AISWSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARD RSATWYYLGLGFDVWGQGTLVTVSS 52-38V.sub.Ldomainaminoacidsequence: (SEQIDNO:6) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQVANVPLTFGQ GTKVEIK

[0028] As will be readily appreciated, the binding specificity of the V.sub.H and V.sub.L domains is dictated by their respective CDR regions, and Table 1 below shows the amino acid sequences for the CDRs in the V.sub.H and V.sub.L domains. Therefore, based on the known CDR sequences, it is contemplated that antibodies and fragments thereof can be prepared that bind CD30 and that include at least some of the CDRs of SEQ ID Nos: 7-24.

TABLE-US-00002 TABLE1 Clone CDR-H1 CDRH-2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 52-2 SYYMH AISWS DRFMP QASQD VASNL QQDAD SEQID GGSTY FIPNT ISNYL ET VPLT NO:7 YADSV LGFDV N SEQID SEQID KG SEQID SEQID NO:11 NO:12 SEQID NO:9 NO:10 NO:8 52-5 SYYMH AISWS DRFMP QASQD DASNL QQDAD SEQID GGSTY FIPNT ISNYL ET VPLT NO:13 YADSV LGFDV N SEQID SEQID KG SEQID SEQID NO:17 NO:18 SEQID NO:15 NO:16 NO:14 52-38 SYWMH AISWS DRSAT QASQD DASNL QQVAN SEQID GDSTY WYYLG ISNYL ET VPLT NO:19 YADSV LGFDV N SEQID SEQID KG SEQID SEQID NO:23 NO:24 SEQID NO:21 NO:22 NO:20

[0029] For example, using the CDRs and V.sub.H and V.sub.L domain information above, IgG.sub.1 antibodies can be prepared. Most typically, but not necessarily, HC and LC with the same preceding numeral (e.g., 64-6) will be present in a CD30 binding antibody. However, other CD30 binding antibodies may have a heavy chain and a light chain with non-identical preceding numeral.

[0030] Of course, it should be appreciated that the inventive subject matter is not limited to the exact sequences noted above, but one or more of the sequences may include one or more amino acid changes. Most preferably, the changes will not result in a substantial reduction of specificity and/or affinity. Thus, contemplated amino acid changes will typically be in the framework regions of the V.sub.H and/or V.sub.L domains, and/or in the constant regions of HC and/or LC. Viewed from a different perspective, amino acid changes will preferably not be present in the CDR region. For example, contemplated sequences will have between 98-99% identity or homology, or between 96-98% identity or homology, or between 92-96% identity or homology, or between 85-92% identity or homology, or between 75-85% identity or homology, most typically (but not necessarily) with the changed amino acids outside the CDRs. Among other options for amino acid changes, one or more amino acids can be changed to humanize a non-human antibody, and/or to move or eliminate one or more glycosylation sites.

[0031] Moreover, it should be noted that contemplated antibodies will expressly include various forms such as monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, single domain antibodies, single-chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), BiKes, and TriKes as is described in more detail below. Of course, it should also be noted that the term antibody expressly includes all classes of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), as well as the corresponding subclasses (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, IgA.sub.2).

[0032] With respect to contemplated antibody fragments it should be noted that fragments will include one or more portions of an antibody that contains CDRs (typically all CDRs of at least one of V.sub.H and V.sub.L), and optionally the framework residues. Thus, antibody fragments will in most cases exhibit an ability to specifically bind to the antigen (here: an epitope of CD30). Among other fragments, especially contemplated fragments include Fab, F(ab).sub.2, Fv, scFv, and mutants thereof, naturally occurring variants, as well as fusion proteins with various non-antibody polypeptides (e.g., toxin, antigen recognition site for a different antigen, enzyme, receptor, receptor ligand, etc.). Viewed from a different perspective, contemplated antibody fragments will have an amino acid sequence of at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0033] In further contemplated aspects, the antibody of fragment thereof may be used for in vitro or in vivo diagnosis and as such be coupled to a detectable label. For example, suitable detectable labels include various enzymes, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable label can be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (e.g., chemical or biological linker) using techniques known in the art. Additionally, or alternatively, contemplated antibodies and fragments thereof may also be coupled to a solid support, which is particularly useful for immunoassays or purification of CD30 or cells expressing CD30. For example, suitable supports include magnetic beads, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, and polypropylene.

[0034] Contemplated antibodies and fragments thereof may also be coupled to or comprise a therapeutic agent to target the agent to a cell expressing CD30. For example, especially contemplated therapeutic agents include chemotherapeutic drugs, radionuclide, and immune stimulants (e.g., cytokine, a cytokine analog, a chemokine, or a checkpoint inhibitor). There are numerous manners of preparing antibody-drug conjugates, and all of these are deemed suitable for use herein.

[0035] In especially preferred aspects, contemplated antibodies or fragments thereof may also be prepared as chimeric proteins in which at least one portion of the antibody is continuous with a second polypeptide (optionally via a preferably flexible linker). For example, suitable chimeric proteins may be configured as chimeric antigen receptors (CAR) that may have an intracellular signaling portion, a transmembrane portion, and an extracellular recognition domain. In such case, it is generally contemplated that the recognition domain includes an antibody fragment (e.g., scFv or single domain) and/or that the intracellular signaling domain comprises an activating/ITAM motif. Among other options, contemplated may be first, second, or third generation CARs with a variety of domains known in the art. For example, suitable CARS will include a CD8 hinge portion, a CD28 transmembrane domain, and a CD3zeta (CD3?) or Fc receptor epsilon (Fc?RI?) signaling domain. Alternatively, the signaling domain may also include one or more of a CD28 signaling domain, a 4-1BB signaling domain, and a CD3zeta (CD3?) signaling domain. Among other options, such chimeric antigen receptors are preferably expressed in cytotoxic immune competent cells, and especially in NK cells and/or T cells.

[0036] On the other hand, and especially where the anti-CD30 antibody or fragment thereof is used to additionally mediate cell or receptor/ligand contact, contemplated chimeric proteins may be constructed as a bispecific fusion protein, as a bispecific killer cell engager (BiKE), or as a trispecific killer cell engager (TriKe). For example, a bispecific fusion protein may comprise the anti-CD30 antibody or portion thereof and a second affinity ligand that selectively binds to a desired target. Such target may be a soluble protein or a cell-bound protein, and especially contemplated targets include PD-L1. On the other hand, contemplated chimeric molecules may be constructed as bispecific polypeptides (e.g., first scFv coupled via linker to second scFv) in which one portion comprises the anti-CD30 antibody or portion thereof and in which the other portion has a binder to a marker specific for an immune competent cell (e.g., anti-CD3?).

[0037] In further contemplated aspects, the anti-CD30 antibody or portion thereof may also be coupled to an IgG-Fc/IL15R?/IL15 hybrid (e.g., ALT803). For example, the anti-CD30 antibody fragment could be a scFv portion that is coupled to one or both arms of the hybrid to so form a TxM (see TxM technology at URL:Altorbioscience.com). Or the anti-CD30 antibody fragment could be a scFv portion that is coupled to one arm of the hybrid, while the other arm of the hybrid could be a scFv portion that binds PD-L1 (or other immune related ligand).

[0038] As should be appreciated, nucleic acids encoding contemplated anti-CD30 antibodies are also expressly considered herein, and the skilled artisan will be readily able to prepare such nucleic acids (e.g., DNA, RNA) and recombinant entities comprising such nucleic acids. Among other options, suitable recombinant entities include yeast, bacterial, and viral expression vectors, linear DNA for genome editing or other integration, RNA, etc. of course, it should be recognized that the recombinant nucleic acids will include suitable regulatory elements to allow for expression of the recombinant construct. Moreover, it should be noted that the nucleic acid will typically make use of codon-optimization with respect to the host cells that include and express the recombinant nucleic acid.

[0039] As will be readily appreciated, use of anti-CD30 antibodies, fragments thereof, or chimeric proteins containing anti-CD30 antibodies or fragments thereof is particularly advantageous where CD30 positive tumor cells are to be targeted, with an anti-CD30 antibody and/or an anti-CD30 CAR on a cytotoxic immune cell. Moreover, where cancer cells express and display CD30, the cells may offer a further therapeutic target (e.g., via targeting with a chimeric molecule that has a CD30 binding portion and an immune stimulatory portion (e.g., ALT-803)).

[0040] In view of these findings, the inventors also contemplate use of various recombinant CD30 binding molecules such as antibodies and fragments thereof as well as cells expressing anti-CD30 CAR molecules and pharmaceutical compositions comprising same. Most typically, such recombinant proteins may be soluble forms of antibodies and fragments thereof, soluble chimeric molecules comprising a CD30 binding portion, or membrane bound molecules such as CAR comprising a CD30 binding portion. For example, recombinant CD30 binding CARs may be expressed in a cytotoxic cell such as a T cell, a natural killer cell, or an NKT cell.

[0041] It is contemplated that such prepared or generated pharmaceutical composition can be administered to a patient having a tumor to increase effectiveness of immune therapy to so treat the tumor (e.g., to modulate (e.g., reduce, abrogate, etc.) immune suppression by the tumor, to reduce the tumor size, etc.). In some embodiments, pharmaceutical composition and/or the tumor vaccine can be administered via systemic injection including subcutaneous, subdermal injection, or intravenous injection. In other embodiments, where the systemic injection may not be efficient (e.g., for brain tumors, etc.) or more localized treatment is desired, it is contemplated that the recombinant immunoglobulin protein complex and/or pharmaceutical compositions can be administered via intratumoral injection. As used herein, the term administering refers to both direct and indirect administration of the compounds and compositions contemplated herein, where direct administration is typically performed by a health care professional (e.g., physician, nurse, etc.), while indirect administration typically includes a step of providing or making the compounds and compositions available to the health care professional for direct administration.

[0042] With respect to dose and schedule of the administration, it is contemplated that the dose and/or schedule may vary depending on depending on the type of protein, protein complex, or the type of the pharmaceutical composition (e.g., virus, bacteria, yeast, in combination with recombinant protein complex, etc.), type and prognosis of disease (e.g., tumor type, size, location), health status of the patient (e.g., including age, gender, etc.). While it may vary, the dose and schedule may be selected and regulated such that the formulation does not provide any significant toxic effect to the host normal cells, yet sufficient to be reduce immune suppression by reduced T cell differentiation and/or activation in the tumor microenvironment. Thus, in a preferred embodiment, an optimal or desired condition of administering the formulation can be determined based on a predetermined threshold. For example, the predetermined threshold may be a predetermined local or systemic concentration of T-cell activating, or T-cell released cytokines (e.g., IL-2, IL-12, IFN-?, IL-12, IL-23, IL-1b, IL-6, or TGF-?, etc.) in the tumor microenvironment. Therefore, administration conditions are typically adjusted to have one or more of those cytokines increased in the tumor microenvironment at least 20%, at least 30%, at least 50%, at least 60%, at least 70% at least for 24 hours, 48 hours, 72 hours, 7 days, etc. Moreover, it is contemplated that the compounds and compositions presented herein may be co-administered (contemporaneously or sequentially) with NK cells. For example, suitable NK cells include autologous NK cells as well as NK92 cells and derivatives thereof (e.g., aNK cells, haNK cells, taNK cells, al commercially available from NantKwest, 9920 Jefferson Blvd. Culver City, CA 90232).

EXAMPLES

[0043] Using the V.sub.H and V.sub.L domains as noted above, the inventors prepared various CD30 binding constructs, and exemplary results for scFv and IgG1 constructs are shown below. For example, Table 2 below shows exemplary results for determination of dissociation constants for scFv and IgG1 constructs using the V.sub.H and V.sub.L domains as noted in the table. More specifically, K.sub.D determination was done by SPR, and average values are shown in ?10.sup.?9 M. Measurements were at 25? C. pH 7.4 with scFv or IgG1 captured on the chip surface, and CD30 was the analyte.

TABLE-US-00003 TABLE 2 aCD30 clone scFv IgG1 52-2 0.8 3.0 52-5 27.1 22.3 52-38 0.77 2.5

[0044] To demonstrate in vitro binding of anti-CD30 constructs to cells expressing CD30, the inventors constructed and used an anti-CD30 IgG1 (52-5) and exposed cells expressing CD30 (here: Jurkat lymphoma cells) to the anti-CD30 IgG1 antibodies, along with a commercially available anti-CD30 antibody. HL-60 cells not expressing CD30 served as negative control. As can be readily seen from the flow cytometry results in FIG. 1, the anti-CD30 IgG1 (52-5) strongly and specifically bound to Jurkat cells and exhibited substantially no binding to HL-60 cells. Moreover, binding of the anti-CD30 IgG1 (52-5) was stronger than the commercially available anti-CD30 antibody. When binding was titrated against Jurkat cells using the anti-CD30 IgG1 (52-38), a dose response curve was obtained as can be seen from FIG. 2 whereas the IgG control afforded no significant binding.

[0045] In a series of further experiments, the inventors constructed an anti-CD30 CAR that included an scFv portion with the V.sub.L and V.sub.H domains as noted above (coupled by a flexible linker portion), and that further included a CD8 hinge portion, a CD28 transmembrane portion, and an Fc receptor epsilon (Fc?RI?) signaling domain. FIG. 3 is a graph depicting exemplary results for CAR specific target cell lysis of CD30 expressing target cells using haNK cells expressing the anti-CD30 CAR constructs as noted and a mock CAR. FIG. 4 is a graph depicting corresponding exemplary results for CAR specific target cell lysis of control target cells not expressing CD30. FIG. 5 is a graph depicting exemplary results for CD30 expression in the target cells of FIGS. 3 and 4, and FIG. 6 is a graph depicting exemplary results for anti-CD30 CAR and mock CAR expression in the haNK cells of FIGS. 3 and 4.

[0046] Notably, the CAR mediated cytotoxicity correlated strictly with the quantity of CD30 present on the target cells as can be seen from FIG. 7. Here, CAR specific target cell lysis of CD30 expressing target cells is shown for cells with low and high expression levels for CD30 as well as a pool of these cells. FIG. 8 exemplarily depicts results for CD30 expression in the target cells of FIG. 7.

[0047] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term about. Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0048] As used herein, the term administering a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, wherein direct administration of the pharmaceutical composition or drug is typically performed by a health care professional (e.g., physician, nurse, etc.), and wherein indirect administration includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should further be noted that the terms prognosing or predicting a condition, a susceptibility for development of a disease, or a response to an intended treatment is meant to cover the act of predicting or the prediction (but not treatment or diagnosis of) the condition, susceptibility and/or response, including the rate of progression, improvement, and/or duration of the condition in a subject.

[0049] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. such as) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0050] As used in the description herein and throughout the claims that follow, the meaning of a, an, and the includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of in includes in and on unless the context clearly dictates otherwise. As also used herein, and unless the context dictates otherwise, the term coupled to is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms coupled to and coupled with are used synonymously.

[0051] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms comprises and comprising should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.