HALF-LIFE EXTENDED IMMTAC BINDING CD3 AND A HLA-A*02 RESTRICTED PEPTIDE

Abstract

The present invention relates to soluble multi-domain binding molecules comprising T cell receptors (TCR) having specificity for an antigen, an immunoglobulin Fc domain or an albumin-binding moiety; and an immune effector domain. Such multi-domain binding molecules are advantageous because they display improved half-life while retaining function.

Claims

1. A multi-domain binding molecule comprising: i) a peptide-major histocompatibility complex (pMHC) binding moiety linked to a T cell engaging immune effector; and ii) a half-life extending domain, comprising an immunoglobulin Fc or an albumin binding domain.

2. A multi-domain binding molecule according to claim 1, wherein the pMHC binding moiety is a T cell receptor (TCR) or TCR-like antibody, comprising TCR and/or antibody variable domains, and at least one constant domain.

3. A multi-domain binding molecule according to claim 2, wherein the TCR is a heterodimeric alpha/beta TCR polypeptide pair.

4. A multi-domain binding molecule according to claim 2, wherein the TCR is a single chain alpha/beta TCR polypeptide.

5. A multi-domain binding molecule according to claim 4, wherein the T-cell engaging immune effector domain is a CD3 effector domain that activates a T cell through interaction with CD3 and or TCR/CD3 complex.

6. A multi-domain binding molecule according to claim 5, wherein the CD3 effector domain comprises an antibody scFv or antibody-like scaffold.

7. A multi-domain binding molecule according to claim 6, wherein the half-life extending domain is an immunoglobulin Fc domain.

8. A multi-domain binding molecule according to claim 6, wherein the half-life extending domain comprises an albumin binding domain.

9. A multi-domain binding molecule according to claim 1, wherein the half-life extending domain is linked to the C or N terminus of the pMHC binding moiety or to the C or N terminus of the T cell engaging immune effector.

10. A multi-domain binding molecule according to claim 1, wherein the half-life extending domain is linked to the pMHC binding moiety or to the T cell engaging immune effector via a linker.

11. A multi-domain binding molecule according to claim 1, for use as a medicament.

12. A pharmaceutical composition comprising the multi-domain binding molecule according to claim 1.

13. Nucleic acid encoding the multi-domain binding molecule according to claim 1.

14. An expression vector comprising nucleic acid of claim 13.

15. A host cell comprising nucleic acid of claim 13, wherein the nucleic acid encoding the multi-domain binding molecule is present as a single open reading frame or two distinct open reading frames encoding the alpha chain and beta chain respectively.

16. A method of making the multi-domain binding molecule according to claim 1 comprising maintaining the host cell of claim 15 under optional conditions for expression of the nucleic acid and isolating the multi-domain antigen binding polypeptide.

17. A method of treatment comprising administering the multi-domain binding molecule of claim 1 to a patient in need thereof.

Description

DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 A) Design of TCR-antiCD3-Fc fusion protein; B) Example sequence of a TCR-antiCD3-Fc fusion protein of the invention FIG. 2 shows that a TCR-antiCD3 fusion incorporating an Fc domain is able to mediate T cell activation in the presence of antigen positive target cells. Data for three IgG1-Fc fusions are shown.

[0082] FIG. 3 A) shows that a TCR-antiCD3 fusion incorporating an albumin binding peptide is able to mediate T cell activation in the presence of antigen positive target cells. Three formats are shown where in the albumin binding peptide is attached to either C-alpha (F1), N-alpha (F2) or C-beta (F3). B) shows that a TCR-antiCD3 fusion incorporating an albumin binding nanobody is able to mediate T cell activation in the presence of antigen positive target cells. Two formats are shown wherein the albumin binding peptide is attached to either C-alpha (R) or C-beta (Y). C) shows that a TCR-antiCD3 fusion incorporating an albumin binding domain antibody (Albudab®) is able to mediate T cell activation in the presence of antigen positive target cells. One format is shown wherein the Albudab® is attached to C-alpha of the TCR-antiCD3 fusion.

[0083] FIG. 4 A) Shows PK characteristics of TCR-antiCD3-Albudab® fusion in mouse serum and B) shows theoretical PK in humans for a TCR-antiCD3-Albudab® fusion based assessment in mouse.

EXAMPLES

[0084] The following examples describe multi-domain binding molecules of the invention, which may be referred to as TCR-antiCD3-Fc fusion proteins or TCR-antiCD3-albumin-binding fusion proteins.

Example 1 (Fc Fusion)

[0085] a) Design of TCR-antiCD3-Fc Fusion Proteins

[0086] In this example, a TCR-antiCD3 fusion protein comprising a high affinity TCR that binds to a HLA-A*02 restricted peptide from PRAME was used. Examples of such molecules are provided in WO2018234319.

[0087] The human IgG1 Fc domain was fused via a linker to the C terminus of the TCR-antiCD3 (see FIG. 1A for schematic). A further two constructs were made comprising functional variants of human IgG1 Fc that are known in the art. Variant 1 does not bind to Fcγ receptors (FcγRs) or complement protein C1q and is therefore functionally silent. Variant 2 shows increased binding to FcRn, which may result in extended in vivo half-life. In each case the Fc domain comprises known knobs in holes mutations Y86T and T22Y to facilitate heterodimerization.

[0088] FIG. 1B shows the sequence of a TCR-anti-CD3-Fc fusion comprising a functionally silent human IgG1 Fc (Variant 1)

[0089] b) Expression and Purification of TCR-antiCD3-Fc Fusion Proteins

[0090] Expression of Fc fusions was performed using a transient expression system based on suspension-adapted Chinese Hamster Ovary (CHO) cells (ExpiCHO Expression system, Thermo Fisher). Cells were transfected according to the manufacturer's instructions, using mammalian expression plasmids containing the relevant TCR chains fused to various Ig Fc domains. Following the harvest, cell culture supernatants were clarified by centrifuging of the cells at 4000-5000×g for 30 minutes in a refrigerated centrifuge. Supernatants were filtered through a 0.22-μm filter and collected for further purification.

[0091] For purification, Fc fusions were first adjusted with buffer before being purified using mAbselect Sure prepacked columns (GE Healthcare or equivalent resins) as per the manufacturer's guidelines. Specific protein containing fractions were pooled and further purified by size exclusion chromatography using appropriate columns (GE Healthcare) in physiologically-relevant buffers. Specific protein containing fractions were pooled and concentrated for downstream testing and storage.

[0092] c) Potent T Cell Activation by TCR-antiCD3-Fc Fusion Proteins

[0093] The TCR-antiCD3-Fc fusion proteins were assessed for their ability to mediate potent redirection of CD3+ T cells against antigen presenting T2 cells. Interferon-γ (IFN-γ) release was used as a read out for T cell activation.

[0094] Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, T2 cells were used as target cells and pulsed with 5 nM PRAME peptide. Target cells were prepared at a density of 1×10.sup.6/ml in assay medium (RPMI 1640 containing 10% heat inactivated FBS and 1% penicillin-streptomycin-L-glutamine) and plated at 50,000 cells per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as effector cells and plated at 35,000 cells per well in a volume of 50 μl. TCR-antiCD3-Fc fusion proteins were titrated to final concentrations of between 10 nM and 0.0001 nM, and added to the well in a volume of 50 μl.

[0095] Plates were prepared according to the manufacturer's instructions. Wells containing target cells, effector cells and fusion proteins were made up to a final volume of 200 μl with assay medium. All reactions were performed in triplicate. Control wells were also prepared with the omission of, either fusion protein, effector cells, or target cells. The plates were incubated overnight (37° C./5% CO.sub.2). The next day the plates were washed three times with wash buffer (1×PBS sachet, containing 0.05% Tween-20, made up in deionised water). Primary detection antibody was then added to each well in a volume of 50 μl. Plates were incubated at room temperature for 2 hours prior to being washed again three times. Secondary detection was performed by adding 50 μl of diluted streptavidin-HRP to each well and incubating at room temperature for 1 hour and the washing step repeated. No more than 15 mins prior to use, one drop (20 μl) of AEC chromogen was added to each 1 ml of AEC substrate and mixed and 50 μl added to each well. Spot development was monitored regularly and plates were washed in tap water to terminate the development reaction. The plates were then allowed to dry at room temperature for at least 2 hours prior to counting the spots using a CTL analyser with Immunospot software (Cellular Technology Limited). Data were prepared and analysed using PRISM software.

[0096] Results presented in FIG. 2 demonstrate that TCR-antiCD3-Fc fusion proteins mediate effective T cell activation in the presence of antigen positive target cells. Ec50 values were in the pM range (238 pM 257 pM and 25 pM respectively for fusions to IgG1-Fc, IgG1-Fc-variant1 and IgG1-Fc-variant2 respectively). Control experiments with antigen negative target cells demonstrated that the functionally silent IgG1-variant2 gave negligible background activity. Functionally silent Fc domains were therefore considered most preferable for therapeutic use.

Example 2 (Albumin Binding)

[0097] a) Design of TCR-antiCD3-Albumin-Binding Fusion Proteins

[0098] In a first design, the TCR-antiCD3 fusion protein comprised a high affinity TCR that binds to a HLA-A*02 restricted peptide from gp100. The amino acid sequence of such molecules is disclosed in WO2011001152. Specifically, the TCR-antiCD3 fusion comprised the alpha chain of SEQ ID No: 45 of WO2011001152, wherein amino acids 1-109 are replaced with SEQ ID No. 8 of WO2011001152, and the amino acid at position 1 is A, based on the numbering of SEQ ID No: 45; and a beta chain of SEQ ID No: 36 of WO2011001152, in which residues 259-370 correspond to SEQ ID No. 27 of WO2011001152, amino acids at position 1 and 2 are A and I respectively. An albumin binding peptide having the amino acid sequence QRLMEDICLPRWGCLWEDDF (as described in Dennis et al., J Biol Chem. 2002 Sep. 20; 277(38):35035-43) was attached to the TCR-antiCD3 fusion via a linker. A suitable linker is GGGGS. Three variants were prepared in which the albumin binding peptide was fused at three different attachment sites: C-alpha (F1), N-alpha (F2) or C-beta (F3).

[0099] In a second design, an albumin binding nanobody was attached to the same TCR-antiCD3 fusion as used in the first design. An albumin binding nanobody having the sequence of SEQ ID No: 52 in WO2006122787 was attached to TCR-antiCD3 fusion via a linker. A suitable linker is GGGGS. Two variants were prepared in which the albumin binding nanobody was fused at two different attachment sites: C-alpha (R) or C-beta (Y).

[0100] In a third design, an albumin binding domain antibody was attached to the C terminus of the TCR-antiCD3 fusion alpha chain. The antibody belongs to the Albudab® platform. Two variants of the domain antibody were used; DOM 7h-10-14 dAb and DOM 7h-11-15 dAb, provided by SEQ ID Nos: 26 and 27 respectively in WO201010893. The antibody was attached via a linker and directly (i.e. without a linker). A suitable linker is GGGGS. The TCR-antiCD3 fusion protein comprised a high affinity TCR that binds to a HLA-A*01 restricted peptide from MAGEA3. Amino acid sequences of such molecules are provided in WO2013041865.

[0101] b) Expression and Purification of TCR-antiCD3-Albumin-Binding Fusion Proteins

[0102] TCR-antiCD3-albumin-binding fusion proteins were expressed in E. coli as inclusion bodies and subsequently refolded and purified, using the same methodology as known in the art for TCR-antiCD3 fusion proteins (for example, see WO2011001152, example 2)

[0103] c) Potent T Cell Activation by TCR-antiCD3-Albumin-Binding Fusion Proteins

[0104] TCR-antiCD3-albumin binding fusion proteins were assessed for their ability to mediate potent redirection of CD3+ T cells against antigen positive cancer cells. Interferon-γ (IFN-γ) release was used as a read out for T cell activation.

[0105] Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, for fusions comprising albumin binding peptide, melanoma Mel526 cells were used as target cells. Target cells were prepared at a density of 1×10.sup.6/ml in assay medium (RPMI 1640, plus 150 μM human serum albumin (HSA) and 1% penicillin-streptomycin-L-glutamine) and plated at 50,000 cells per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as effector cells and plated at 30,000 cells per well in a volume of 50 μl. TCR-antiCD3-Fc fusion proteins were titrated to final concentrations of between 10 nM, and 0.0001 nM, and added to the well in a volume of 50 μl.

[0106] For fusions comprising Albudab®, myeloma EJM cells were used as target cells. Target cells were prepared at a density of 1×10.sup.6/ml in assay medium (RPMI 1640, plus 45 μM HSA, and 1% penicillin-streptomycin-L-glutamine) and plated at 50,000 cells per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as effector cells and plated at 30,000 cells per well in a volume of 50 μl. TCR-antiCD3-Fc fusion proteins were titrated to final concentrations of between 10 nM, and 0.0001 nM, and added to the well in a volume of 50 μl.

[0107] Plates were prepared and developed as described in Example 1c

[0108] FIG. 3A-C shows that fusion of an albumin binding moiety to a TCR-antiCD3 fusion protein, mediates effective T cell activation in the presence of antigen positive target cells. Ec50 values were in the pM range (137.4/178.0/137.1)

Example 3 (Extended Half-Life)

[0109] a) PK Assessment of TCR-antiCD3-Albumin-Binding Fusion Proteins

[0110] The PK characteristics of TCR-antiCD3-AlbudAb fusions were investigated in mouse serum.

[0111] Mice were dose with 0.1 mg/kg of fusion protein by intravenous bolus injection and serum samples were taken at regular intervals over a period of 120 hours. PK assessment was carried out by using an ELISA based assay. In brief, biotinylated pHLA complex was attached to streptavidin-coated plates and serum samples were then added. A detection step was carried out using a primary goat antibody against anti-CD3 scFv and a HRP-conjugated anti-goat IgG activated for colourimetric detection with TMB at 450 nm. Results generated were used to confirm the presence and binding activity of the TCR-antiCD3-Albudab fusion by using a dilution series and analysis against a standard curve. The results are reported as % activity and used to generate a plot of Cmax over time.

[0112] The resulting PK data are shown in FIG. 4A. Note that the samples denoted ‘TCR-Alb (10-14)’ and ‘TCR-Alb (11-15)’ refer to the TCR-antiCD3 fusions described in Example 2a. ‘TCR-Alb (D)’ is a control sample fused to a non-albumin binding Albudab, and ‘TCR’ refers to the TCR-antiCD3 without Albudab.

[0113] The PK data from FIG. 4A were used to calculate a theoretical PK profile for TCR-antiCD3-AlbudAb fusions in humans (FIG. 4B). Fusion to Albudab is predicted to extend in-vivo half life from 7 h to 264 h.