CD3-specific binding molecules

Abstract

The present invention relates to specific binding molecules which bind to CD3, particularly antibodies and fragments thereof, with improved properties.

Claims

1. An antibody or antibody fragment thereof that specifically binds to CD3, comprising an immunoglobulin variable light chain (VL) domain and an immunoglobulin variable heavy chain (VH) domain, wherein the immunoglobin VL domain comprises the sequence: TABLE-US-00011 (SEQIDNO:16) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIK; and the immunoglobulin VH domain comprises the sequence: TABLE-US-00012 (SEQIDNO:18) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVAL INPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSS; or (SEQIDNO:19) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVAL INPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSS.

2. The antibody or antibody fragment thereof of claim 1, comprising a single chain variable fragment (scFv) molecule.

3. The antibody or antibody fragment thereof of claim 1, wherein the immunoglobin VL domain and the immunoglobulin VH domain are connected via a linker.

4. A fusion polypeptide comprising: i) a targeting moiety selected from a T cell receptor (TCR), an antibody, or an antibody fragment; and ii) the antibody or antibody fragment thereof of claim 1.

5. The fusion polypeptide of claim 4, wherein the TCR is a heterodimeric alpha/beta TCR polypeptide pair or a single chain alpha/beta TCR polypeptide.

6. The fusion polypeptide of claim 4, wherein the TCR comprises a non-native disulfide bond between the constant region of the alpha chain and the constant region of the beta chain.

7. The fusion polypeptide of claim 4, wherein the antibody or antibody fragment thereof is fused to the C terminus or the N terminus of the targeting moiety, optionally via a linker.

8. A pharmaceutical composition comprising the antibody or antibody fragment thereof of claim 1.

9. A nucleic acid molecule encoding the antibody or antibody fragment thereof of claim 1.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. An isolated host cell comprising the expression vector of claim 10, wherein the nucleic acid encoding the antibody or antibody fragment thereof is present as a single open reading frame or two distinct open reading frames.

12. A method of making an antibody or antigen binding fragment thereof that specifically binds to CD3, comprising culturing the isolated host cell of claim 11 under conditions for expression of a nucleic acid encoding the antibody or antibody fragment thereof and isolating the antibody or antibody fragment thereof.

13. The antibody or antibody fragment thereof of claim 1, wherein the immunoglobin VL domain comprises the sequence: TABLE-US-00013 (SEQIDNO:16) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIK; and the immunoglobin VH domain comprises the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYK GVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSS (SEQ ID NO: 19).

14. The antibody or antibody fragment thereof of claim 2, wherein the scFv molecule has the sequence: TABLE-US-00014 (SEQIDNO:21) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSS.

15. The antibody or antibody fragment thereof of claim 1, wherein the immunoglobin VL domain comprises the sequence: TABLE-US-00015 (SEQIDNO:16) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIK; and the immunoglobin VH domain comprises the sequence: TABLE-US-00016 (SEQIDNO:18) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVA LINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCAR SGYYGDSDWYFDVWGQGTLVTVSS.

16. The antibody or antibody fragment thereof of claim 2, wherein the scFv molecule has the sequence: TABLE-US-00017 (SEQIDNO:20) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSS.

17. An antibody or antigen binding fragment thereof that specifically binds to CD3 comprising an immunoglobulin VL domain comprising Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, and VLCDR3, and an immunoglobulin VH domain comprising CDRs VHCDR1, VHCDR2, and VHCDR3, wherein the amino acid sequence of; TABLE-US-00018 VLCDR1is (SEQIDNO:1) QDIRNY; VLCDR2is YTS; VLCDR3is (SEQIDNO:2) QQGNTLPWT; VHCDR1is (SEQIDNO:3) GYSFTGYA; VHCDR2is (SEQIDNO:4) INPYKGVS;and VHCDR3is (SEQIDNO:5) ARSGYYGDSDWYFDV.

18. A heterodimeric TCR-anti-CD3 antibody fusion molecule, comprising: (a) a first polypeptide chain that comprises a TCR alpha chain variable domain; and (b) a second polypeptide chain that comprises a TCR beta chain variable domain fused to an anti-CD3 scFv having the amino acid sequence: TABLE-US-00019 (SEQIDNO:20) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSS;or (SEQIDNO:21) AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSS.

19. The antibody or antibody fragment thereof of claim 3, wherein the linker comprises between 5 amino acid residues to 30 amino acid residues.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1provides VH and VL amino acid sequences of improved UCHT1 variants. CDRs are underlined. Mutations are shown in bold. FIG. 1 contains SEQ ID NOS 16 and 18-19, respectively, in order of appearance.

(2) FIG. 2provides example amino acid sequences a TCR-anti-CD3 fusion protein, incorporating improved anti-CD3 scFv variants. FIG. 2 contains SEQ ID NOS 34 and 32-33, respectively, in order of appearance.

(3) FIG. 3demonstrates a TCR-CD3 fusion incorporating improved anti-CD3 scFv variant 1 has a better therapeutic window relative to non-mutated anti-CD3 (A) and a TCR-CD3 fusion incorporating improved anti-CD3 scFv variant 2 has a higher Emax relative to non-mutated anti-CD3 (B) FIG. 4demonstrates improved T cell killing properties mediated by a TCR-CD3 fusion incorporating UCHT1 variant 1 and variant 2.

(4) The invention is further described in the following non-limiting examples.

EXAMPLES

(5) The following examples describe bifunctional binding molecules of the invention, which may be referred to as TCR-antiCD3-bispecific proteins.

(6) 1) Preparation of TCR-Anti-CD3 Bispecific Fusion Proteins with Improved Anti-CD3

(7) Fusion proteins comprising a TCR and an anti-CD3 scFv are known in the art (for example, see WO2011001152, WO2017109496, WO2017175006 and WO2018234319). These molecules comprise a humanised UCHT1 scFv fragment.

(8) In this example, variants of a TCR-anti-CD3 fusion protein described in WO2018234319 were produced that incorporate either anti-CD3 variant 1 (T165A) or anti-CD3 variant 2 (T165A+I202F). Mutations were introduced using standard mutagenesis and cloning methods (such as described in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Amino acid sequences of VL and VH for T165A and T165A+I202F are provided in FIG. 1. Amino acids sequences of a TCR-anti-CD3 incorporating the sequences provided in FIG. 1 are provided in FIG. 2.

(9) TCR-anti-CD3 fusion proteins comprising T165A, T165A+I202F or non-mutated (WT) UCHT1 scFv were expressed in E. coli as inclusion bodies and subsequently refolded and purified using the methods described in WO2018234319, example 2)

(10) 2) Increased Therapeutic Window and Maximum T Cell Activation Mediated by TCR-and-CD3 with Variants T165A and T165A+I202F

(11) A) IFN- Release

(12) The TCR-anti-CD3 fusion proteins described above were assessed for their ability to mediate activation of CD3+ T cells in the presence of antigen positive and antigen negative cells. Interferon- (IFN-) release was used as a read out for T cell activation.

(13) Assays were performed using a human IFN- ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, Me1624 melanoma cells were used as antigen positive target cells. Granta-519 B cell lymphoma cells were used as antigen negative target cells. Target cells were prepared at a density of 110.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 l. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as CD3+ effector cells and plated at 35,000 cells per well in a volume of 50 l. TCR-anti-CD3 proteins were titrated to final concentrations of between 10 nM and 0.0001 nM, and added to the well in a volume of 50 l.

(14) Plates were prepared and developed according to the manufacturer's instructions. Wells containing target cells, effector cells and fusion proteins were made up to a final volume of 200 l 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 (1PBS 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 l. Plates were incubated at room temperature for 2 hours prior to being washed again three times. Secondary detection was performed by adding 50 l 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 l) of AEC chromogen was added to each 1 ml of AEC substrate and mixed and 50 l 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.

(15) The therapeutic window was calculated by determining the relative potency of T cell activation mediated by TCR-antiCD3 variant and WT, against antigen-positive cells, and comparing Ec50 values after curve-fitting in Prism. For antigen-negative cells robust Ec50 values could not be obtained, so a minimal cross-reactive concentration was determined by setting a threshold number of spots (eg. 25 spots per well), and identifying by interpolation the concentration that would first exceed that number.

Results

(16) T165A

(17) To obtain a robust determination of therapeutic window, data were averaged from 4 Ag+ plates and 4 Ag-plates each of which had non-mutated and T165A side-by-side. FIG. 3A shows the curves obtained from one plate. Data for each of the individual plates is shown in the tables below.

(18) TABLE-US-00010 Plate 1 Plate 2 Plate 5 Plate 6 Target cells Mel 624 (Ag+) Mel 624 (Ag+) Mel 624 (Ag+) Mel 624 (Ag+) Effector cells Donor 1 Donor 1 Donor 2 Donor 2 T165A EC50 116 pM 137 pM 160 pM 446 pM Wt EC50 62.2 pM 60.9 pM 79.3 pM 72.1 pM T165A relative 0.54 0.44 0.50 0.16 potency Plate 3 Plate 4 Plate 7 Plate 8 Target cells Granta 519 (Ag) Granta 519 (Ag) Granta 519 (Ag) Granta 519 (Ag) Effector cells Donor 1 Donor 1 Donor 2 Donor 2 T165A cross-reactive 1.56 nM 7.16 nM 0.698 nM 0.615 nM concentration Wt cross-reactive 0.165 nM 0.811 nM 0.206 nM 0.152 nM concentration T165A relative 0.11 0.11 0.29 0.25 cross-reactivity

(19) The relative potency against antigen positive cells (i.e. Ec50 of WT divided by Ec50 of T165A) gave an average of 0.41. The relative cross-reactivity against antigen negative cells (i.e. conc of WT giving 25 spots/concentration of T165A giving 25 spots) gave an average of 0.19. These data demonstrate that the therapeutic window of T165A is approximately 2 greater than the window for the WT.

(20) T165A+I202F

(21) FIG. 3B shows that the T165A+I202F variant results in a higher maximum T cell activation (Emax) relative to WT. In this case the therapeutic window is similar to WT. These data demonstrate that T165A+I202F is more efficient at activating T cells.

(22) B) T Cell Mediated Killing

(23) The ability of TCR-anti-CD3 fusion proteins to mediate redirected T cell killing of antigen positive and antigen negative tumour cells was investigated using the IncuCyte platform (Essen BioScience). This assay allows real time detection by microscopy of the release of Caspase-317, a marker for apoptosis.

Method

(24) Assays were performed using the CellPlayer 96-well Caspase-3/7 apoptosis assay kit (Essen BioScience, Cat. No. 4440) and carried out according the manufacturers protocol. Briefly, target cells (Me1624 (antigen positive) or MDA MB 231 (antigen negative) cells were plated at 10,000 cells per well and incubated overnight to allow them to adhere. TCR-anti-CD3 fusion proteins were added at concentrations between 0.05 nM and 0.0125 nM (for antigen positive cells) and between 20 nM and 0.125 nM (for antigen negative cells). CD3+ effector cells (PBMC) were used at an effector target cell ratio of 10:1 (100,000 cells per well). NucView assay reagent was made up at 30 M and 25 l added to every well and the final volume brought to 150 l (giving 5 M final conc). The plate was placed in the IncuCyte instrument and images at regular intervals over 3 to 5 days. The number of apoptotic cells in each image was determined and recorded as object count per mm.sup.2. Assays were performed in triplicate. Graphs were prepared using PRISM software.

Results

(25) The resulting killing curves for WT, T165A and T165A+I202F against antigen positive and antigen negative cells are shown in FIG. 4. Note that for clarity, only the curves for the indicated concentrations are shown.

(26) T165A

(27) T165A shows a small reduction in T cell killing of antigen positive cells relative to WT at a concentration of 0.005 nM. Killing of antigen negative cells is not observed up to 20 nM whereas for WT killing is observed at a concentration of 1 nM. These data confirm the above findings that T165A has an improved therapeutic window.

(28) T165A+I202F

(29) T165A+I202F shows increase in T cell mediated killing at a given concentration (e.g. 0.005 nM) against antigen positive cells. Killing of antigen negative cells is comparable to WT. These data confirm the above findings and demonstrate that T165A+I202F is more efficient at activating T cells.

(30) The data presented in the example demonstrate that TCR-antiCD3 fusion proteins incorporating T165A or T165A+I202F UCHT1 variants have improved therapeutic properties