Anti-CD3 antibodies and uses thereof

Abstract

The present invention relates to anti-CD3 antibodies and uses thereof. The present invention relates to antibodies that specifically bind CD3. The present invention relates to antibodies that specifically bind to PSMA. The present invention relates to antibodies that specifically bind to CD3 and PSMA. The present invention relates to antibodies that specifically bind to IL1RAP. The present invention relates to antibodies that specifically bind to CD33. The present invention relates to antibodies that specifically bind to CD3 and IL1RAP. The present invention relates to antibodies that specifically bind to CD3 and CD33. The present invention relates to antibodies that specifically bind to TMEFF2. The present invention relates to antibodies that specifically bind to CD3 and TMEFF2. The invention relates to fragments of the antibodies, polynucleotides encoding the antibodies or fragments thereof, and methods of making and using the antibodies.

Description

[0001] This application is a divisional application of the invention application with the application date of May 21, 2019, the Chinese national application number of 201980049271.7, and the invention title of "Anti-CD3 Antibodies and Their Uses". Technical Field

[0002] The disclosure provided herein relates to anti-CD3 antibodies and antigen-binding fragments thereof that are capable of specifically binding to human and non-human cluster of differentiation 3 (CD3), and specifically to anti-CD3 antibodies and antigen-binding fragments that cross-react with CD3 of non-human mammals (e.g., cynomolgus monkeys); PSMA antibodies and antigen-binding fragments thereof that are capable of specifically binding to human and non-human prostate-specific membrane antigen (PSMA); IL1RAP antibodies and antigen-binding fragments thereof that are capable of specifically binding to human and non-human IL1RAP; CD33 antibodies and antigen-binding fragments thereof that are capable of specifically binding to human and non-human CD33; and bispecific antibodies that are capable of specifically binding to CD3; PSMA; IL1RAP; CD33; CD3 and PSMA; CD3 and IL1RAP; or CD3 and CD33. The present disclosure also relates to the use of such antibodies and antigen-binding fragments in the treatment of cancer, autoimmune and / or inflammatory diseases, and other disorders. Background Art

[0003] Bispecific antibodies and antibody fragments have been explored as a means to recruit cytotoxic T cells to kill tumor cells. However, the clinical use of many bispecific antibodies that recruit T cells is limited by challenges including adverse pharmacokinetics, potential immunogenicity, and manufacturing issues. Thus, there is a great need for bispecific antibodies that recruit cytotoxic T cells to kill tumor cells with reduced toxicity and favorable manufacturing characteristics.

[0004] The human CD3 T cell antigen receptor protein complex consists of six different chains: one CD3γ chain (SwissProt P09693), one CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and one CD3ζ chain homodimer (SwissProt P20963) (εγ:εδ:ζζ), which homodimer associates with the T cell receptor α and β chains. This complex plays an important role in coupling antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for the immune response. Summary of the Invention

[0005] The present disclosure provides a isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: a heavy chain and a light chain, the heavy chain comprising: a heavy chain complementarity-determining region (HCDR) 1 comprising SEQ ID NO:662, an HCDR2 comprising SEQ ID NO:663 and an HCDR3 comprising SEQ ID NO:664, the light chain comprising: a light chain complementarity-determining region (LCDR) 1 comprising SEQ ID NO:671, an LCDR2 comprising SEQ ID NO:673 and an LCDR3 comprising SEQ ID NO:690; a heavy chain variable region comprising SEQ ID NO:652 and a light chain variable region comprising SEQ ID NO:661; a heavy chain comprising SEQ ID NO:640 and a light chain comprising SEQ ID NO:676; or comprising: a heavy chain and a light chain, the heavy chain comprising: an HCDR1 comprising SEQ ID NO:662, an HCDR2 comprising SEQ ID NO:663 and an HCDR3 comprising SEQ ID NO:664, the light chain comprising: an LCDR1 comprising SEQ ID NO:773, an LCDR2 comprising SEQ ID NO:673 and an LCDR3 comprising SEQ ID NO:690; a heavy chain variable region comprising SEQ ID NO:657 and a light chain variable region comprising SEQ ID NO:678; or a heavy chain comprising SEQ ID NO:675 and a light chain comprising SEQ ID NO:678.

[0006] Also provided is an isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof that specifically binds cynomolgus macaque (Macaca fascicularis) or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of about 300 nM or less.

[0007] In some embodiments, the isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof has one, two, three, or four of the following properties:

[0008] · Binds human and cynomolgus macaque CD3+ T lymphocytes with a calculated EC50 of 20 nM or less, and binds HEK cells expressing cynomolgus macaque CD3 with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding to CD3+ T lymphocytes and binding to HEK cells expressing cynomolgus macaque CD3 is less than 5-fold, and wherein the calculated EC50 is measured by flow cytometry in a whole-cell binding assay at 0 °C;

[0009] · Binds recombinant CD3d from human with an equilibrium dissociation constant (KD) of 12 nM or less

[0010] (SEQ ID NO:691), or in combination with recombinant human CD3e (SEQ ID NO:

[0011] 636), or recombinant cynomolgus macaque CD3d (SEQ ID NO:692), where KD was measured using a Proteon surface plasmon resonance ProteOn XPR36 system at

[0012] +25 °C;

[0013] · Binds to residues 1-6 of CD3e, as determined by X-ray crystallography; or

[0014] · Activates T cells or induces CD69 expression to a level similar to that of cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0015] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID No:662, 663, 664, 671, 673, and 690, respectively; VH and VL that are SEQ ID NO:652 and 661, respectively; or HC and LC that are SEQ ID NO:640 and 676, respectively.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID No:662, 663, 664, 773, 673, and 690, respectively; VH and VL that are SEQ ID NO:657 and 678, respectively, or HC and LC that are SEQ ID NO:675 and 677, respectively.

[0017] Also described herein are bispecific antibodies that comprise a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen, wherein the first domain comprises an antibody or antigen-binding fragment thereof described herein.

[0018] The bispecific antibodies described herein may comprise a first domain that comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 670, respectively; and a second domain that comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:697, 683, 698, 699, 792, and 686, respectively;

[0019] The bispecific antibodies described herein may comprise a first domain comprising VH and VL that are SEQ ID NO: 652 and 661, respectively; and a second domain comprising VH and VL that are SEQ ID NO: 681 and 682, respectively.

[0020] The bispecific antibodies described herein may comprise a first domain comprising HC and LC that are SEQ ID NO: 640 and 676, respectively; and a second domain comprising HC and LC that are SEQ ID NO: 679 and 680, respectively.

[0021] The bispecific antibodies described herein may comprise a first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 671, 673, and 670, respectively; and a second domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 704, 705, 706, 707, 708, and 709, respectively.

[0022] The bispecific antibodies described herein may comprise a first domain comprising VH and VL that are SEQ ID NO: 652 and 661, respectively; and a second domain comprising VH and VL that are SEQ ID NO: 702 and 703, respectively.

[0023] The bispecific antibodies described herein may comprise a first domain comprising HC and LC that are SEQ ID NO: 640 and 676, respectively; and a second domain comprising HC and LC that are SEQ ID NO: 700 and 701, respectively.

[0024] The present disclosure also provides an isolated bispecific CD3×PSMA antibody comprising a first domain that binds to cells expressing recombinant cynomolgus macaque or human CD3d or CD3e with an affinity of 300 nM or less, wherein the binding to the cells is measured by flow cytometry; and a second domain that specifically binds to PSMA.

[0025] In some embodiments, the bispecific CD3×PSMA antibody comprises a first domain that:

[0026] · Binds to human and cynomolgus macaque CD3+ T lymphocytes with a calculated EC50 of 20 nM or less, and binds to HEK cells expressing cynomolgus macaque CD3 with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding to CD3+ T lymphocytes and binding to HEK cells expressing cynomolgus macaque CD3 is less than 5-fold, and wherein the calculated EC50 is measured by flow cytometry in a whole cell binding assay at 0 °C;

[0027] · Binds to recombinant CD3d from human with an equilibrium dissociation constant (KD) of 12 nM or less

[0028] (SEQ ID NO:691), or binds to recombinant CD3e from human (SEQ ID NO:

[0029] 636), or binds to recombinant CD3d from cynomolgus macaque (SEQ ID NO:692), or binds to recombinant CD3e from cynomolgus macaque (SEQ ID NO:693), wherein the KD is measured using a Proteon surface plasmon resonance assay on a ProteOn XPR36 system at +25 °C

[0030] ;

[0031] · As detected by peptide mapping, shows no methionine or tryptophan oxidation, or shows no asparagine deamidation, or shows no asparagine isomerization;

[0032] · Binds to residues 1-6 of CD3e, as determined by X-ray crystallography; or

[0033] · Activates T cells or induces CD69 expression to a level similar to that of cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0034] The present invention also describes a pharmaceutical composition comprising the antibody described herein and a pharmaceutically acceptable carrier.

[0035] The present disclosure also provides methods for generating the antibodies described herein, including culturing a host cell comprising a vector under conditions for expressing the antibody, and recovering the antibody produced by the host cell, wherein the vector comprises a polynucleotide encoding the antibody described herein or an antigen-binding fragment thereof. The method for generating a bispecific CD3×PSMA antibody may include mixing a monospecific bivalent CD3 antibody having two identical HC1s and two identical LC1s with a monospecific bivalent PSMA antibody having two identical HC2s and two identical LC2s in a mixture at a molar ratio of about 1:1; introducing a reducing agent into the mixture; incubating the mixture for about ninety minutes to about six hours; removing the reducing agent; and purifying the bispecific CD3×PSMA antibody comprising HC1, LC1, HC2, and LC2.

[0036] The invention also describes a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody for a time sufficient to treat the cancer.

[0037] The present disclosure also provides a kit comprising the antibody described herein. The kit may further include reagents for detecting the antibody and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention will be further understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the antibodies and methods of the present disclosure, exemplary embodiments of the antibodies and methods are shown in the drawings; however, the antibodies and methods are not limited to the specific embodiments disclosed. In the drawings:

[0039] Figure 1A and Figure 1B shows an anti-CD3 antibody produced in OmniRat. The VH ( Figure 1A ) and VL ( Figure 1B ) sequences of the active anti-CD3 mAb produced in OmniRat were aligned with the human germline sequences from IMGT. The CDR regions are underlined. Sequence differences are shown in bold. Figure 1A SEQ ID NO:651, 651, 653, 656, 655, 20, 654, and 717 are disclosed, and Figure 1B SEQ ID NO:658, 688, 660, 659, 659, 659, 659, and 718 are disclosed, all in the order of appearance, respectively.

[0040] Figure 2 shows a cell-based binding assay for evaluating the binding ability of individual rat hybridoma supernatants to human purified CD3+ T lymphocytes.

[0041] Figure 3Shows a cell-based binding assay for evaluating the binding ability of individual rat hybridoma supernatants to cynomolgus monkey purified CD3+ T lymphocytes.

[0042] Figure 4 Shows the results of a competition assay of the hybridoma supernatant, which evaluates the ability of the hybridoma supernatant to compete with the commercially available anti-human CD3 antibody SP34-2, which has a known epitope and cross-reacts with cynomolgus monkey CD3.

[0043] Figure 5 Shows a representative binding curve of the anti-CD3 antibody on primary human T cells.

[0044] Figure 6 Shows a representative competitive binding curve of the anti-CD3 antibody conjugated with AlexaFluor 488 SP34-2 on primary human T cells.

[0045] Figure 7 Shows the binding of the light chain (LC)-engineered BLW-2E6 mAb to primary human T cells.

[0046] Figure 8 Shows the binding of the heavy chain (HC)-engineered BLW-2E6 to primary human T cells.

[0047] Figure 9 Shows AlexaFluor TM (AF)488 CD3 saturation in human T cells by FACS analysis. The mean fluorescence intensity values collected were plotted as a function of antibody molecule concentration. The Kd value for each donor was derived and the mean value was obtained. The saturation binding constant (KdT) for human T cells was derived as 5.6 ± 1.0 nM (n = 4) and was used herein to determine the Kd binding affinity. One human donor was excluded because it did not meet the minimum survival rate criterion of at least 60% during the analysis. The "LS" identifier in the legend refers to individual human T cell donors.

[0048] Figure 10 Shows the inhibition curves of the bivalent anti-CD3 antibodies CD3B376 and CD3B450 competing for binding to the AlexaFluor488 SP-34 anti-CD3 antibody. The IC50 values were derived as 29 nM and 60 nM, respectively.

[0049] Figure 11 Shows the sensorgram of the BLW-2E6 variant binding to hCD3ε(1-27)-Tn25.

[0050] Figures 12A to 12E Shows the anti-CD3 antibody CD3B376 determined by DSC ( Figure 12A )、CD3B450(Figure 12B )、Thermal stability of CD3B389( Figure 12C )、CD3B467( Figure 12D ), and the overlay of the thermal profiles of all candidates( Figure 12E ).

[0051] Figures 13A to 13B Shows the comparison of the thermal stability of the overlay of the anti-CD3 antibody thermal profiles of CD3B376 and CD3B389( Figure 13A ); the overlay of the thermal profiles of CD3B450 and CD3B467( Figure 13B ).

[0052] Figure 14 Shows the LNCAP cell binding of a subset of affinity matured PSMA×CD3 bispecific antibodies.

[0053] Figure 15 Shows the LNCAP cell binding of a subset of affinity matured PSMA×CD3 bispecific antibodies.

[0054] Figure 16 Shows the PSMA-negative PC3 cell binding results of affinity matured PSMA×CD3 bispecific antibodies.

[0055] Figure 17 Shows the results of PSMA×CD3 affinity matured bispecific Abs in a functional cell killing assay.

[0056] Figure 18 Shows the antibody-antigen interaction in the CD3B334:CD3 complex. CD3 residues are in the ellipse and CD3B334 residues are in the box.

[0057] Figure 19 Shows an assay based on primary human and cynomolgus monkey T cells, which is used to determine the ability of hybridoma hits to activate T cells, as measured by CD69 activation.

[0058] Figure 20 Shows the anti-tumor efficacy of PS3B79 in the LnCAP AR.TB human prostate xenograft in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice a week and the results are expressed as mean tumor volume, in mm 3 ±SEM (*, p < 0.0001).

[0059] Figure 21Shows the anti-tumor efficacy of PS3B90 in the LnCAP AR.TB human prostate xenograft in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice a week, and the results are presented as mean tumor volume, in mm 3 ±SEM (*, p < 0.001).

[0060] Figures 22A to 22D Shows the titration curves of anti-PSMA phage panning hits binding to human LNCaP cells. Figure 22A Shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 22B Shows the titration curves of M52 and M110; Figure 22C Shows the titration curves of M85, M87, and M81; and Figure 22D Shows the titration curves of M52 and M84. In Figure 22D , Fab expression in the mammalian-expressed supernatant was normalized by octet, and titration was performed against human LNCAP, PSMG5 (cynomolgus monkey PSMA HEK), or PSMG9 (chimpanzee PSMA HEK) cells using flow cytometry. The relationship between geometric mean fluorescence intensity (GeoMFI) and Fab concentration was plotted using GraphPad Prizm.

[0061] Figures 23A to 23D Shows the titration curves of anti-PSMA phage panning hits binding to HEK cells expressing chimpanzee PSMA. (Titration curves of mammalian Fab supernatants of anti-PSMA phage panning hits against chimpanzee PSMA HEK). Figure 23A Shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 23B Shows the titration curves of M52 and M110; Figure 23C Shows the titration curves of M81, M52, M85, and M87; and Figure 23D Shows the titration curves of M52 and M84. In Figure 23D , Fab expression in the mammalian-expressed supernatant was normalized by octet, and titration was performed against human LNCAP, PSMG5 (cynomolgus monkey PSMA HEK), or PSMG9 (chimpanzee PSMA HEK) cells using flow cytometry. The relationship between geometric mean fluorescence intensity (GeoMFI) and Fab concentration was plotted using GraphPad Prizm.

[0062] Figures 24A to 24DShows the titration curve of anti-PSMA phage panning hits that bind to HEK cells expressing cynomolgus PSMA. (Mammalian Fab supernatant titration curve of anti-PSMA phage panning hits against cynomolgus PSMA HEK). Figure 24A Shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 24B Shows the titration curves of M52 and M110; Figure 24C Shows the titration curves of M81, M52, M85, and M87; and Figure 24D Shows the titration curves of M52 and M84. In Figure 24D Fab expression was normalized for mammalian-expressed supernatants by octet, and titrations were performed against human LNCAP, PSMG5 (cynomolgus PSMA HEK), or PSMG9 (chimpanzee PSMA HEK) cells using flow cytometry. The geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm.

[0063] Figure 25 Shows the overall structure of PSMM84 Fab that binds to the human PSMA ECD homodimer.

[0064] Figure 26 Shows a close-up view of the major interaction of PSMA with the light chain of PSMB83.

[0065] Figure 27 Shows a close-up view of the major interaction of PSMA with the heavy chain of PSMB83.

[0066] Figure 28 Shows a comparison of the epitope residues of PSMB83 within the sequences of human (SEQ ID NO:719), mouse (SEQ ID NO:720), and cynomolgus (cyno) (SEQ ID NO:721) PSMA. The epitope residues are shaded, and the sequence differences are underlined.

[0067] Figure 29 Shows the complementary residues of PSMB83. The CDRs are underlined, and the complementary residues are shaded. Figure 29 SEQ ID NOs: 722 - 727 are disclosed in appearance order, respectively.

[0068] Figure 30 Shows the interaction map of direct contacts between PSMA and PSMM84. Van der Waals interactions are shown as dashed lines, and H bonds are solid arrows pointing to the backbone atoms.

[0069] Figure 31 Shows the expression levels of anti-PSMA Fab clones derived from PSMM84 compared to the expression of the parental PSMB83. The raw luminescence counts were plotted against the log concentration.

[0070] Figure 32 Shows the binding of anti-PSMA Fab clones derived from PSMB83 to human PSMA compared to the binding of the parental PSMM84. The raw luminescence counts were plotted against the log concentration.

[0071] Figure 33 Shows the binding of anti-PSMA Fab clones derived from PSMB83 to cynomolgus monkey PSMA compared to the binding of the parental PSMB83. The raw luminescence counts were plotted against the log concentration.

[0072] Figure 34 Shows the in vitro mediated T cell cytotoxicity of IC3B19 and IC3B34 against LAMA-84 cells in whole blood after 48 hours. The concentrations of IC3B19 and IC3B34 are provided in the table at the bottom of the figure.

[0073] Figure 35 Shows the in vitro mediated T cell activation of IC3B19 and IC3B34 in whole blood after 48 hours. The concentrations of IC3B19 and IC3B34 are provided in the table at the bottom of the figure.

[0074] Figures 36 to 55 show the binding of IC3B19 and IC3B34 mediated T cells to IL1RAP+ target cell line LAMA-84 (endogenous and exogenously added tumor cells). The cytotoxicity of 10 pro-inflammatory cytokines from the supernatant of whole blood (n = 15 donors) and the T cell activation assay with the addition of exogenously added LAMA-84 IL1RAP+ tumor cell line were evaluated. For these figures, statistically significant differences are shown in bold text.

[0075] Figures 36A to 36B Shows the T cell IL-1β release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 36A ) and the corresponding 4PL regression parameter estimates ( Figure 36B ).

[0076] Figures 37A to 37B Shows the T cell IL-1β release mediated by IC3B19 and IC3B34 after 48 hours ( Figure 37A ) and the corresponding 4PL regression parameter estimates ( Figure 37B ).

[0077] Figures 38A to 38B Shows the T cell IL-2 release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 38A) and the corresponding 4PL regression parameter estimates ( Figure 38B )

[0078] Figures 39A to 39B shows the T cell IL-2 release mediated by IC3B19 and IC3B34 after 48 hours ( Figure 39A ) and the corresponding 4PL regression parameter estimates ( Figure 39B )

[0079] Figures 40A to 40B shows the T cell IL-4 release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 40A ) and the corresponding 4PL regression parameter estimates ( Figure 40B )

[0080] Figure 41 shows the T cell IL-4 release mediated by IC3B19 and IC3B34 after 48 hours.

[0081] Figures 42A to 42B shows the T cell IL-6 release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 42A ) and the corresponding 4PL regression parameter estimates ( Figure 42B )

[0082] Figure 43 shows the T cell IL-6 release mediated by IC3B19 and IC3B34 after 48 hours.

[0083] Figure 44 shows the T cell IL-8 release mediated by IC3B19 and IC3B34 after 24 hours.

[0084] Figure 45 shows the T cell IL-8 release mediated by IC3B19 and IC3B34 after 48 hours.

[0085] Figures 46A to 46B shows the T cell IL-10 release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 46A ) and the corresponding 4PL regression parameter estimates ( Figure 46B )

[0086] Figures 47A to 47B shows the T cell IL-10 release mediated by IC3B19 and IC3B34 after 48 hours ( Figure 47A ) and the corresponding 4PL regression parameter estimates ( Figure 47B )

[0087] Figure 48 shows the T cell IL-12p70 release mediated by IC3B19 and IC3B34 after 24 hours.

[0088] Figure 49 Shows T cell IL-12p70 release mediated by IC3B19 and IC3B34 after 48 hours.

[0089] Figure 50 Shows T cell IL-13 release mediated by IC3B19 and IC3B34 after 24 hours.

[0090] Figures 51A to 51B Shows T cell IL-13 release mediated by IC3B19 and IC3B34 after 48 hours ( Figure 51A ) and the corresponding 4PL regression parameter estimates ( Figure 51B ).

[0091] Figures 52A to 52B Shows T cell IFN-γ release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 52A ) and the corresponding 4PL regression parameter estimates ( Figure 52B ).

[0092] Figures 53A to 53B Shows T cell IFN-γ release mediated by IC3B19 and IC3B34 after 48 hours ( Figure 53A ) and the corresponding 4PL regression parameter estimates ( Figure 53B ).

[0093] Figures 54A to 54B Shows T cell TNF-α release mediated by IC3B19 and IC3B34 after 24 hours ( Figure 54A ) and the corresponding 4PL regression parameter estimates ( Figure 54B ).

[0094] Figures 55A to 55B Shows T cell TNF-α release mediated by IC3B19 and IC3B34 after 48 hours ( Figure 55A ) and the corresponding 4PL regression parameter estimates ( Figure 55B ).

[0095] Figure 56 Shows that IC3B19 and IC3B34, rather than bispecific antibodies with blank arms (IAPB57×B23B49 or B23B39×CD3B219), induce target-specific cytotoxicity in NCI-H1975 cells. In this assay, the cytotoxic EC50 varies threefold between IC3B19 and IC3B34, with values of 0.018 nM and 0.057 nM, respectively.

[0096] Figure 57Shows the anti-tumor efficacy of IAPB57×CD3B376 in H1975 human NSCLC xenografts in T cell humanized NSG mice. Subcutaneous H1975 tumors were measured twice a week, and the results are presented as mean tumor volume, expressed as mm3±SEM, *p<0.0001.

[0097] Figure 58 Shows the comparison of the isoelectric points of various anti-PSMA constructs.

[0098] Figure 59 Plots the change in wavelength compared to the CNTO5825 control molecule. Control CNTO607 exhibits characteristic strong self-interaction. Error bars represent the standard deviation of triplicate measurements.

[0099] Figure 60 Shows the comparison of retention times on IgG columns and control columns in the anti-PSMA cross-interaction assay.

[0100] Figures 61A to 61B Shows the ex vivo evaluation of the cytotoxicity and T cell activation of blasts in fresh AML patient whole blood using CD33×CD3 bispecific antibodies with anti-CD3 arms CD3B219 and CD3B376. Figure 61A Shows the percentage of total cytotoxicity of AML cells using CD33 bispecific antibodies or CD3×blank control. Figure 61B Shows the T cell activation induced by CD33 bispecific antibodies or CD3×blank control. No Fc blocker was added.

[0101] Figures 62A to 62C Shows the CD33×CD3 T cell-mediated cytotoxicity assay. The CD33×CD3 bispecific antibody using anti-CD3 arms CD3B219 and anti-CD3B376 was incubated with human pan T cells and AML cell lines that are wild type (KG1, Figure 62A ) for the CD33 SNP rs12459419 mutation, heterozygous (SH2, Figure 62B ) or homozygous (OCIAML3, Figure 62C ). After incubation at 37°C and 5% CO2 for 48 hours, the cytotoxicity of total tumor cells was measured by flow cytometry.

[0102] Figures 63A to 63B Shows the ex vivo evaluation of the cytotoxicity of C33B904 antibody paired with CD3B219 or CD3B376 against MOLM-13 cells exogenously added to normal healthy human whole blood (N = 6 donors): After 48 hours using CD33×CD3 bispecific antibodies and the corresponding blank×CD3 control, the percentage of cytotoxicity of MOLM-13 cells ( Figure 63A) and CD33 + CD14 + Cytotoxicity percentage of monocytes ( Figure 63B ).

[0103] Figures 64A to 64B Ex vivo evaluation of the cytotoxicity and T cell activation of monocytes in fresh whole blood from six normal cynomolgus monkey donors using the CD33×CD3 bispecific antibodies with anti-CD3 arms CD3B219 and CD3B376. Figure 64A Shows the CD33 of the CD33 bispecific antibody or its CD3×blank control + CD14 + Total cytotoxicity percentage of cynomolgus monkey monocytes. Figure 64B Shows the T cell activation induced by the CD33 bispecific antibody or its CD3×blank control. No Fc blocker was added.

[0104] Figure 65 Shows the anti-tumor efficacy of C3CB189 in the MOLM-13 human AML xenograft in T cell humanized NSG mice. The disseminated MOLM-13 tumors were imaged by bioluminescence (BLI) twice a week, and the results are presented as mean radiance (p / s / cm 2 / sr) ± SEM (n = 8 - 10 / group). *p ≤ 0.0001 for treatment vs. control, calculated by two-way ANOVA with Bonferroni test.

[0105] Figure 66 Shows the survival rate of animals treated with C3CB189 in the MOLM-13 human AML xenograft in T cell humanized NSG mice. The survival rate of mice carrying MOLM-13 was graphically represented using the Kaplan-Meier curve and evaluated by the log-rank (Mantel-Cox) test. *p ≤ 0.0001 for the treatment group vs. the control group.

[0106] Figure 67 Shows the anti-tumor efficacy of C3CB88 in the MOLM-13 human AML xenograft in T cell humanized NSG mice. The disseminated MOLM-13 tumors were imaged by bioluminescence (BLI) twice a week, and the results are presented as mean radiance (p / s / cm 2 / sr) ± SEM (n = 8 - 10 / group). *p ≤ 0.0001 for treatment vs. control, calculated by two-way ANOVA with Bonferroni test.

[0107] Figure 68Shows the survival rate of MOLM-13 human AML xenografts in T cell-humanized NSG mice in animals treated with C3CB88. The survival rate of mice carrying MOLM-13 was graphically represented using Kaplan-Meier curves and evaluated by log-rank (Mantel-Cox) test. *p ≤ 0.05 for the treatment group compared to the control group.

[0108] Figure 69 Shows the alignment of selected anti-TMEFF2 antibody heavy chain variable regions (VH). The VH regions are identified by their SEQ ID NO: at the start of each line.

[0109] Figure 70 Shows the alignment of selected anti-TMEFF2 antibody light chain variable regions (VL). The VH regions are identified by their SEQ ID NO: at the start of each line.

[0110] Figure 71 Shows the reduction in mean tumor volume per mouse treated with 0.5 mg / kg TMCB132 in an ex vivo LnCaP prostate cancer model in male NGS mice.

[0111] Figure 72 Shows the efficacy of TMEB762×CD3B376 in an established LNCaP xenograft in T cell-humanized NSG mice.

[0112] Figure 73 Shows T cell activation in LnCaP prostate cancer cells in response to the administration of TMCB132.

[0113] Figure 74 Shows the T cell-mediated cytotoxicity of TMCB132.

[0114] Figure 75 Shows the anti-tumor efficacy of TMCB132 in T cell-humanized mice. Detailed Description

[0115] All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if fully set forth herein.

[0116] It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0117] Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, exemplary materials and methods are described herein. When describing and claiming the present invention, the following terms will be used.

[0118] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of two or more cells and the like.

[0119] "Specifically bind", "specifically binds" or "binds" means that an antibody binds to an antigen or an epitope within that antigen with a higher affinity than to other antigens. Typically, an antibody binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (K -8 ) of about 5×10 -9 M or less (e.g., about 1×10 -10 M or less, about 1×10 -11 M or less, about 1×10 -12 M or less or about 1×10 D ), and typically this K D is at most one percent of the K D for the antibody binding to a non-specific antigen (such as BSA, casein). Standard procedures can be used to measure the dissociation constant. However, an antibody that specifically binds to an antigen or an epitope within the antigen may have cross-reactivity to other related antigens, for example, cross-reactivity to the same antigen from other species (homologous) (such as human or monkey, e.g., cynomolgus (cyno), Pan troglodytes (chimpanzee, chimp)). While a monospecific antibody specifically binds to one antigen or one epitope, a bispecific antibody specifically binds to two different antigens or two different epitopes.

[0120] "Antibody" broadly refers to and includes immunoglobulin molecules, specifically including monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding sites with the desired specificity. "Full-length antibody molecule" includes two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as their multimers (such as IgM). Each heavy chain consists of a heavy-chain variable region (VH) and a heavy-chain constant region (composed of the domains CH1, hinge, CH2, and CH3). Each light chain consists of a light-chain variable region (VL) and a light-chain constant region (CL). The VH region and VL region can be further subdivided into hypervariable regions, which are called complementarity-determining regions (CDR) and are interspersed with framework regions (FR). Each VH and VL consists of three CDRs and four FR segments and are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0121] "Complementary determining region (CDR)" is the antibody region that binds antigen. CDRs can be defined using various delineations, such as Kabat (Wu et al., 1970, J Exp Med, Vol. 132, pp. 211-250) (Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., 1987, J Mol Biol, Vol. 196, pp. 901-917), IMGT (Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77), and AbM (Martin and Thornton, 1996, J Mol Biol, Vol. 263, pp. 800-815). The correspondence between various delineations and variable region numbering is described (see, e.g., Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77; Honegger and Pluckthun, 2001, J Mol Biol, Vol. 309, pp. 657-670; International Immunogenetics (IMGT) database; web resource, http: / / www_imgt_org). Programs (such as abYsis from UCL Business PLC) can be used to delineate CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT, or AbM).

[0122] Immunoglobulins can be assigned to five main classes based on the amino acid sequence of the heavy chain constant domain, namely IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of their constant domains, the light chains of antibodies from any vertebrate species can be assigned to one of two completely different types, namely κ and λ.

[0123] "Antigen-binding fragment" refers to the antigen-binding portion of an immunoglobulin molecule. Antigen-binding fragments can be synthetic, enzymatically obtainable, or genetically engineered polypeptides, and include VH, VL, VH and VL, Fab, F(ab')2, Fd and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of an antibody (such as FR3-CDR3-FR4 portions, HCDR1, HCDR2 and / or HCDR3, and LCDR1, LCDR2 and / or LCDR3). VH and VL domains can be joined together via a synthetic linker to form various types of single-chain antibody designs, where in cases where the VH and VL domains are expressed by separate single-chain antibody constructs, the VH / VL domains can pair intra- or intermolecularly to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or a bivalent antibody; as described, for example, in International Patent Publications WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047.

[0124] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known variations (such as removal of the C-terminal lysine from the antibody heavy chain) or post-translational modifications (such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation). Monoclonal antibodies typically bind to one epitope. Bispecific monoclonal antibodies bind to two different epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific or multispecific, such as bispecific monovalent, bivalent or multivalent.

[0125] "Isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds an antigen and is substantially free of antibodies that specifically bind antigens other than that antigen). In the case of a bispecific CD3 antibody, the bispecific antibody specifically binds both CD3 and a second antigen. "Isolated antibody" encompasses antibodies isolated to a higher purity, such as antibodies of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0126] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. The humanized antibody may contain substitutions in the framework such that the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0127] A "human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, then that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is derived from a system using human germline immunoglobulin or rearranged immunoglobulin genes, then the human antibody contains a heavy chain variable region and a light chain variable region that are "derived from" human-derived sequences. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Due to differences between the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations, or the intentional introduction of substitutions into the framework or CDRs, or both, "human antibodies" typically contain amino acid differences compared to immunoglobulins expressed in humans. Generally, the amino acid sequence of a "human antibody" has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may contain a consensus framework sequence derived from the analysis of human framework sequences (such as that described in Knappik et al., 2000, J Mol Biol, Vol. 296, pp. 57-86); or a synthetic HCDR3 incorporated into a human immunoglobulin gene library displayed on phage (such as that described in Shi et al., 2010, J Mol Biol, Vol. 397, pp. 385-396 and International Patent Publication WO2009 / 085462).

[0128] "Percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The alignment for purposes of determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein, the amino acid sequence identity % values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed in the U.S. Copyright Office (Washington, D.C., 20559) in a user documentation under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. When using ALIGN-2 for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be stated as the given amino acid sequence A has or contains a certain percent amino acid sequence identity to the given amino acid sequence B) is calculated as follows:

[0129] 100 times the fraction X / Y,

[0130] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B is not equal to the percent amino acid sequence identity of B to A. Unless otherwise specifically stated, all percent amino acid sequence identity % values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0131] An antibody in which the antigen binding site is derived from a non-human species is not included in the definition of a "human antibody."

[0132] "Recombinant" refers to DNA, antibodies, and other proteins that are prepared, expressed, formed, or isolated by recombinant means when fragments from different sources are joined to produce recombinant DNA, antibodies, or proteins.

[0133] "Epitope" refers to the part of an antigen that specifically binds to an antibody. An epitope typically consists of chemically reactive (such as polar, nonpolar, or hydrophobic) surface groups such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural features as well as specific charge characteristics. An epitope can be composed of contiguous and / or non-contiguous amino acids that form a conformational space unit. For a discontinuous epitope, amino acids from different parts of the linear sequence of the antigen are brought close together in three-dimensional space due to the folding of the protein molecule. The antibody "epitope" depends on the method used to identify the epitope.

[0134] "Paratope" refers to the part of an antibody that specifically binds to an antigen. A paratope can be linear in nature or can be discontinuous, formed by the spatial relationship between non-adjacent amino acids of the antibody rather than a linear series of amino acids. "Light chain paratope" and "heavy chain paratope" or "light chain paratope amino acid residues" and "heavy chain paratope amino acid residues" refer respectively to the antibody light chain and heavy chain residues that contact the antigen, or generally, "antibody paratope residues" refer to those antibody amino acids that contact the antigen.

[0135] "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. A bispecific antibody may have cross-reactivity with other related antigens, for example, cross-reactivity with the same antigen from other species (homologs) such as humans or monkeys, e.g., cynomolgus (cyno) or chimpanzee, or may bind to epitopes shared between two or more different antigens.

[0136] "Multispecific" refers to an antibody that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. A multispecific antibody may have cross-reactivity with other related antigens, for example, cross-reactivity with the same antigen from other species (homologs) such as humans or monkeys, e.g., cynomolgus (cyno) or chimpanzee, or may bind to epitopes shared between two or more different antigens.

[0137] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications (such as one or more substitutions, insertions, or deletions).

[0138] "Vector" means a polynucleotide capable of replicating within a biological system or of moving between such systems. Vector polynucleotides typically contain elements such as an origin of replication, a promoter, a polyadenylation signal, or a selectable marker, the function of which is to facilitate the replication or maintenance of these polynucleotides within a biological system, such as a cell, virus, animal, plant, or recombinant biological system that utilizes biological components capable of replicating the vector. The vector polynucleotide can be a single-stranded or double-stranded DNA or RNA molecule, or a hybrid molecule of these molecules.

[0139] "Expression vector" means a vector that can be used to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector within a biological system or a reconstituted biological system.

[0140] "Polynucleotide" means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical means. Double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA, are typical examples of polynucleotides. "Polynucleotide" can refer to a synthetic molecule comprising a chain of nucleotides covalently linked by a phosphosugar backbone or other equivalent covalent chemical. cDNA is an exemplary synthetic polynucleotide.

[0141] "Polypeptide" or "protein" means a molecule comprising at least two amino acid residues joined by peptide bonds to form a polypeptide. Small polypeptides of fewer than 50 amino acids can be referred to as "peptides".

[0142] "Flow cytometry" is a technique used to analyze the physical and chemical properties of particles in a fluid as they pass through at least one laser. Cellular components are fluorescently labeled and then excited by the laser to emit light of different wavelengths (Adan et al., Critical Reviews in Biotechnology, 2016, pages 1549 - 7801).

[0143] "Anti-idiotype (anti-Id) antibody" means an antibody that recognizes an antigenic determinant (e.g., paratope or CDR) of an antibody. Methods for producing or preparing anti-idiotype antibodies are well known in the art. (Lathey, J. et al., Immunology, 1986, volume 57, number 1, pages 29 - 35). Anti-Id antibodies can be antigen-blocking or non-blocking. Antigen-blocking anti-Id antibodies can be used to detect free antibodies in a sample (e.g., CD3). Non-blocking anti-Id antibodies can be used to detect total antibodies in a sample (free, partially bound to antigen, or fully bound to antigen antibodies). Anti-Id antibodies can be prepared by immunizing an animal, in which the anti-Id antibody is being prepared, with an antibody. In some embodiments described herein, anti-idiotype antibodies are used to detect the level of a therapeutic antibody in a sample.

[0144] An anti-Id antibody can also be used as an immunogen to induce an immune response in another animal, thereby generating a so-called anti-anti-Id antibody. The anti-anti-Id antibody can be identical to the original mAb that induced the anti-Id antibody in terms of the epitope. Thus, by using an antibody against the idiotypic determinant of a monoclonal antibody, other clones expressing antibodies of the same specificity can be identified. The anti-Id antibody can be altered (thereby generating anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein for anti-CD3 antibodies.

[0145] PSMA refers to prostate-specific membrane antigen. The amino acid sequence of chimpanzee (also known as chimpanzee or chimp) PSMA is shown in SEQ ID NO:49. The extracellular domain spans residues 44 - 750 of SEQ ID NO:49, the transmembrane domain spans residues 20 - 43, and the cytoplasmic domain spans residues 1 - 19. The amino acid sequence of cynomolgus monkey (also known as cynomolgus monkey, macaque or cyno) PSMA is shown in SEQ ID NO:50. The extracellular domain spans residues 44 - 750 of SEQ ID NO:50, the transmembrane domain spans residues 20 - 43, and the cytoplasmic domain spans residues 1 - 19. The amino acid sequence of human PSMA is shown in SEQ ID NO:51. The extracellular domain spans residues 44 - 750 of SEQ ID NO:51, the transmembrane domain spans residues 20 - 43, and the cytoplasmic domain spans residues 1 - 19.

[0146] Throughout the specification, "CD3-specific" or "specifically binds to CD3" or "anti-CD3 antibody" refers to an antibody that specifically binds to the CD3-ε polypeptide (SEQ ID NO:635), including an antibody that specifically binds to the extracellular domain (ECD) of CD3-ε (SEQ ID NO:636). CD3-ε forms the T cell receptor-CD3 complex together with CD3-γ, -δ and -ζ, and the T cell receptor α / β and γ / δ heterodimers. This complex plays an important role in coupling antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for the immune response.

[0147] SEQ ID NO:635 (human CD3ε)

[0148] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0149] SEQ ID NO:636 (extracellular domain of human CD3ε)

[0150] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0151] As used herein, the terms "interleukin-1 receptor accessory protein", "IL1RAP", and "IL1-RAP" specifically include the human IL1RAP protein, e.g., those described in GenBank accession number AAB84059, NCBI reference sequence: NP_002173.1, and UniProtKB / Swiss-Prot accession number Q9NPH3-1 (see also Huang et al., 1997, Proc. Natl. Acad. Sci. USA. Vol. 94, No. 24, pp. 12829-12832). IL1RAP has also been referred to in the scientific literature as IL1 R3, C3orf13, FLJ37788, IL-1RAcP, and EG3556.

[0152] "CD33" refers to the cluster of differentiation 33 protein. CD33 is a single-pass transmembrane glycoprotein of 67 kilodaltons (kD) and is a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family. CD33 is primarily considered a myeloid differentiation antigen, being expressed at low levels in myeloid progenitors, neutrophils, and macrophages, and highly expressed in circulating monocytes and dendritic cells. The extracellular domain of human CD33 (Uniprot P20138) (SEQ ID NO: 636) and cynomolgus monkey CD33 (XP_005590138.1) are examples of proteins used in generating CD33-specific antibodies according to the present disclosure.

[0153] "TMEFF2" refers to the human transmembrane protein with EGF-like and two follistatin-like domains 2, also known as tomoregulin 2. The amino acid sequence of full-length human TMEFF2 is shown in SEQ ID NO:77. The extracellular domain of TMEFF2 is shown in SEQ ID NO:575 and spans residues 40-374 of full-length TMEFF2. The TMEFF2 extracellular domain carries three distinct subdomains: Kazal-like 1 (residues 85-137), Kazal-like 2 (residues 176-229), and the EGF domain (residues 261-301). The TMEFF2 EGF domain is shown in SEQ ID NO:577. The TMEFF2 "membrane-proximal region" refers to the TMEFF2 region of SEQ ID NO:629, which encompasses the EGF domain and the N-C terminal linker region (e.g., residues 230-320 of full-length human TMEFF2 of SEQ ID NO:77). Unless explicitly indicated as from a non-human species, all references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the corresponding protein, polypeptide, or protein fragment. Thus, unless indicated as from a non-human species, such as "mouse TMEFF2" or "monkey TMEFF2", etc., "TMEFF2" means human TMEFF2.

[0154] SEQ ID NO:77 (full-length human TMEFF2)

[0155] MVLWESPRQCSSWTLCEGFCWLLLLPVMLLIVARPVKLAAFPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0156] SEQ ID NO:575 (extracellular domain of human TMEFF2)

[0157] FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0158] TMEFF2 EGF domain SEQ ID NO:577

[0159] HHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCE

[0160] TMEFF2 membrane-proximal region SEQ ID NO:629

[0161] NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0162] "TMEFF2-positive cancer" refers to cancer tissue or cancer cells that exhibit a measurable level of TMEFF2 protein. The level of TMEFF2 protein can be measured on live cells or lysed cells using well-known assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay. "Overexpress (Overexpress, overexpressed, and overexpressing)" refers interchangeably to a sample that has a measurable higher level of tumor antigen compared to a reference sample, such as cancer cells, malignant cells, or cancer tissue. Overexpression can be caused by gene amplification or by increased transcription or translation. The expression and overexpression of a protein in a sample can be measured on live cells or lysed cells using well-known assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay. The expression and overexpression of a polynucleotide in a sample can be measured, for example, using fluorescence in situ hybridization, Southern blot, or PCR techniques. A protein or polynucleotide is overexpressed when the level of the protein or polynucleotide in the sample is at least 1.5 times the level of the protein or polynucleotide in the reference sample. The selection of the reference sample is well-known.

[0163] "Sample" refers to a collection of fluid-like, cellular, or tissue-like material isolated from a subject, as well as fluid, cells, or tissue present within a subject. Exemplary samples are biological fluids, such as blood, serum, and serous fluid, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal, and other body cavities, fluid collected by bronchoalveolar lavage, liquid solutions in contact with a subject or biological source such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, tumor tissue obtained by fine needle aspiration or surgical resection.

[0164] As used herein, "cancer cell" or "tumor cell" refers to cancerous, pre-cancerous, or transformed cells in vivo, ex vivo, or in tissue culture that have undergone a phenotypic change, either spontaneously or induced. These changes do not necessarily involve the uptake of new genetic material. While transformation can occur by infection with a transforming virus and the incorporation of new genomic nucleic acid, or the uptake of exogenous nucleic acid, it can also occur spontaneously or after exposure to a carcinogen, resulting in an endogenous gene mutation. Examples of transformation / cancer are morphological changes, cell immortality, abnormal growth control, focus formation, proliferation, malignancy, regulation of tumor-specific marker levels, invasion, and tumor growth in vitro, in vivo, and ex vivo in a suitable animal host (such as nude mice, etc.) (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed., 1994)).

[0165] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, should be understood to be modified in all instances by the term "about". Thus, numerical values typically include the recited value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, numerical ranges used expressly include all possible sub-ranges, all individual numerical values within the range, including integers within such ranges and fractions within such ranges.

[0166] "Effector antigen" is an antigen from cells of the immune system that can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, and / or cytokine release. Such effector antigens are from, for example but not limited to, T cells and natural killer (NK) cells. Examples of suitable specificities for effector antigens include but are not limited to CD3 or CD3 subunits, such as CD3ε for T cells and CD16 for NK cells. Such cell surface molecules of effector cells are suitable for mediating cell killing. An effector cell is a cell of the immune system that can be stimulated or triggered to exhibit cytotoxicity, phagocytosis, antigen presentation, and / or cytokine release. Such effector cells are, for example but not limited to, T cells, natural killer (NK) cells, granulocytes, monocytes, macrophages, dendritic cells, and antigen-presenting cells. Examples of suitable specificities for effector cells include but are not limited to CD2, CD3, and CD3 subunits, such as CD3e, CD5, CD28, and other components of the T cell receptor (TCR) for T cells; CD16, CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336, (NKp44), CD337 (NKp30), NKp80, NKG2C, and NKG2D, DNAM, NCR for NK cells; CD18, CD64, and CD89 for granulocytes; CD18, CD32, CD64, CD89, and mannose receptor for monocytes and macrophages; CD64 and mannose receptor for dendritic cells; and CD35. In certain embodiments of the invention, those specificities of effector cells, i.e., cell surface molecules, are suitable for mediating cell killing when a bispecific or multispecific molecule binds to such cell surface molecules, thereby inducing cell lysis or apoptosis.

[0167] "Bispecific CD3 antibody" refers to a molecule that includes at least one binding domain that specifically binds to CD3 and at least one binding domain that specifically binds to a second antigen, such as a bispecific antibody that includes a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. The domains that specifically bind to CD3 and the second antigen are typically V H / V L Yes. A bispecific CD3 antibody can be monovalent with respect to its binding to CD3 or the second antigen. In some embodiments, the second antigen or target antigen is a cell surface antigen expressed on a target cell other than an immune effector cell. In some embodiments, the second antigen is a tumor-associated antigen (TAA). Exemplary TAAs are PSMA, CD33, IL1RAP, and TMEFF2.

[0168] "Bispecific PSMA×CD3 antibody", "PSMA / CD3 antibody", "bispecific anti-PSMA×CD3 antibody", or "anti-PSMA / CD3 antibody", etc., refer to a molecule that includes at least one binding domain that specifically binds to PSMA and at least one binding domain that specifically binds to CD3. The domains that specifically bind to PSMA and CD3 are typically V H / V L Yes. A bispecific anti-PSMA×CD3 antibody can be monovalent with respect to its binding to PSMA or CD3.

[0169] "Bispecific CD33×CD3 antibody", "CD33 / CD3 antibody", "bispecific anti-CD33×CD3 antibody", or "anti-CD33 / CD3 antibody", etc., refer to a molecule that includes at least one binding domain that specifically binds to CD33 and at least one binding domain that specifically binds to CD3. The domains that specifically bind to CD33 and CD3 are typically V H / V L Yes. A bispecific anti-CD33×CD3 antibody can be monovalent with respect to its binding to CD33 or CD3.

[0170] "Bispecific IL1RAP×CD3 antibody", "IL1RAP / CD3 antibody", "bispecific anti-IL1RAP×CD3 antibody", or "anti-IL1RAP / CD3 antibody", etc., refer to a molecule that includes at least one binding domain that specifically binds to IL1RAP and at least one binding domain that specifically binds to CD3. The domains that specifically bind to IL1RAP and CD3 are typically V H / V L Yes. A bispecific anti-IL1RAP×CD3 antibody can be monovalent with respect to its binding to IL1RAP or CD3.

[0171] "Bispecific anti-TMEFF2 / anti-CD3 antibody", "TMEFF2 / CD3 antibody", "TMEFF2xCD3 antibody", etc. refer to antibodies that bind to TMEFF2 and CD3.

[0172] "Valency" refers to the specified number of binding sites specific for an antigen present in a molecule. Thus, the terms "monovalent", "divalent", "tetravalent", and "hexavalent" refer to one, two, four, and six binding sites specific for an antigen present in a molecule, respectively. "Multivalent" refers to two or more binding sites specific for an antigen present in a molecule.

[0173] "Antigen-specific CD4+ or CD8+ T cells" refer to CD4+ or CD8+ T cells activated by a specific antigen or its immunostimulatory epitope.

[0174] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" may be used interchangeably.

[0175] Unless otherwise explicitly stated, throughout the specification, amino acid residues in the antibody constant regions are numbered according to the EU index, as described in Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0176] Table 1. Conventional single-letter and three-letter amino acid codes used herein

[0177] Amino acid Three-letter code Single-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Gln E Glutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile I Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Yes Valine Val V

[0178] Composition of substances

[0179] The present invention provides anti-CD3 antibodies and antigen-binding fragments thereof, antibodies specifically binding to PSMA and antigen-binding fragments thereof, antibodies specifically binding to CD33 and antigen-binding fragments thereof, antibodies specifically binding to IL1RAP and antigen-binding fragments thereof, multispecific antibodies comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen, and multispecific antibodies specifically binding to CD3 and one or more of PSMA, CD33, IL1RAP, and TMEFF2. The present invention provides polypeptides and polynucleotides encoding the antibodies of the present invention or their complementary nucleic acids, vectors, host cells, and methods for their preparation and use.

[0180] General aspects of the antibodies described herein

[0181] The anti-CD3 antibodies or antigen-binding fragments described herein include variants having one or more amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the anti-CD3 antibody or antigen-binding fragment. In the context of the present invention, unless otherwise specified, the following symbols are used to describe mutations: i) substitution of an amino acid at a given position is written, for example, as K409R, which means that lysine at position 409 is replaced by arginine; and ii) for a particular variant, any amino acid residue is indicated using a specific three-letter or one-letter code (including the codes Xaa and X). Thus, replacement of lysine at position 409 with arginine is represented as: K409R, or replacement of lysine at position 409 with any amino acid residue is represented as K409X. In the case of deletion of lysine at position 409, it is represented as K409*. Variants having one or more amino acid substitutions, deletions, or additions can be prepared by those skilled in the art.

[0182] These variants can include: (a) variants in which one or more amino acid residues are replaced with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include substituents, and (d) variants in which the polypeptide is fused to another peptide or polypeptide (such as a fusion partner, protein tag, or other chemical moiety), which can confer useful properties on the polypeptide, such as, for example, an epitope of an antibody, a polyhistidine sequence, a biotin moiety, etc. The antibodies or antigen-binding fragments described herein can include variants in which amino acid residues from one species are replaced with corresponding residues in another species at conservative or non-conservative positions. In other embodiments, amino acid residues at non-conservative positions are replaced with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (deletion, mutation, etc.), chemical techniques, and enzymatic techniques, are known to those of ordinary skill in the art.

[0183] The antibodies or antigen-binding fragments described herein can be of the IgM, IgD, IgG, IgA, or IgE isotype. In some embodiments, the antibody isotype is of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody is of the IgG1 isotype. In some embodiments, the antibody is of the IgG2 isotype. In some embodiments, the antibody is of the IgG3 isotype. In some embodiments, the antibody is of the IgG4 isotype. The specific part of an antibody or its antigen-binding fragment is determined by the amino acid sequence and arrangement of the CDRs. Thus, the CDRs of one isotype can be converted to another isotype without changing antigen specificity. Thus, such antibody isotypes are within the scope of the antibody or antigen-binding fragment.

[0184] Human IgG isotypes are divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. They share greater than 95% homology in the amino acid sequences of the CH1, CH2, and CH3 regions, but the main differences are shown in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region binds to the Fc receptor (FcγR) on the surface of immune effector cells such as natural killer cells and macrophages, resulting in the lysis of target cells. In CDC, the antibody mediates target cell killing by triggering the complement cascade on the cell surface. In ADCP, the antibody mediates the elimination of antibody-coated target cells through the internalization by phagocytic cells such as macrophages or dendritic cells. The antibodies described herein include antibodies having the described characteristics with variable domains in combination with any IgG isotype, including modified forms in which the Fc region has been modified to modulate various effector functions.

[0185] For many applications of therapeutic antibodies, Fc-mediated effector functions are undesirable because they may potentially pose a safety hazard due to cell depletion. Modulating effector functions can be achieved by engineering the Fc region to weaken its binding to FcγR or complement factors. The binding of IgG to the activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb), FcγR or the first component of complement (C1q) depends on residues located in the hinge region and CH2 domain. Mutations can be introduced in IgG1, IgG2, and IgG4 to reduce or silence Fc-mediated effector functions. The antibodies described herein may include these modified forms.

[0186] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies comprise an engineered Fc region having one or more of the following properties: (a) reduced effector function compared to the parental Fc; (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb, and / or FcγRIIIa; (c) reduced affinity for FcγRI; (d) reduced affinity for FcγRIIa; (e) reduced affinity for FcγRIIIb; or (f) reduced affinity for FcγRIIIa.

[0187] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are, for example, of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments where the antibody has an IgG4 isotype, the antibody contains S228P, F234A, and L235A substitutions in its Fc region compared to wild-type IgG4. In some embodiments where the antibody has an IgG1 isotype, the antibody contains L234A and L235A substitutions in its Fc region. The antibodies described herein may include these modified forms.

[0188] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are of the IgG4 isotype, optionally comprising the heavy chain substitution S228P.

[0189] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are of the IgG1 isotype and optionally contain heavy chain substitutions L234A, G237A, P238S, H268A, A330S, and P331S when compared to wild-type IgG1.

[0190] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are of the IgG2 isotype and optionally contain heavy chain substitutions L234A, G237A, P238S, H268A, V309L, A330S, and P331S when compared to wild-type IgG2.

[0191] Wild-type IgG4

[0192] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:602)

[0193] Wild-type IgG1

[0194] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

[0195] SGVHTFPAVLQSS

[0196] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

[0197] TCPPCPAPELLGG

[0198] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:601)

[0199] Wild-type IgG2

[0200] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT

[0201] SGVHTFPAVLQSS

[0202] GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:711)

[0203] In certain embodiments, labeled anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are provided. Exemplary labels or moieties for direct detection (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radiolabels) and labels and moieties for indirect detection (e.g., via enzyme reactions or molecular interactions) (e.g., enzymes or ligands). Exemplary labels include radiolabels (e.g., 32 P, 14 C, 111 I, 125 I, 3 H, 131 I), fluorescent labels such as 649, epitope tags, biotin, chromophore labels, ECL labels, or enzymes. More specifically, the labels include ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorescein dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, dyes, etc.

[0204] In addition to the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the antibodies and antigen-binding fragments are provided. Vectors containing the polynucleotides are also provided, as well as cells expressing the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies or antigen-binding fragments provided herein. Cells capable of expressing the disclosed vectors are also described. These cells can be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as Escherichia coli). The antibodies can also be produced by hybridoma cells.

[0205] Generation of monospecific antibodies

[0206] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies of the invention are human.

[0207] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies of the invention are humanized.

[0208] The monospecific antibodies of the invention described herein (e.g., anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies) can be generated using a variety of techniques. For example, the hybridoma method described in Kohler and Milstein, Nature, Vol. 256, p. 495, 1975 can be used to generate monoclonal antibodies. In the hybridoma method, a human, chimpanzee, or rhesus macaque PSMA, CD33, IL1RAP, TMEFF2, or CD3, or a fragment of PSMA, CD33, IL1RAP, TMEFF2, or CD3 (such as the extracellular domain of PSMA, CD33, IL1RAP, TMEFF2, or CD3) is used to immunize a mouse or other host animal (such as a hamster, rat, or monkey), and then spleen cells from the immunized animal are fused with myeloma cells using standard methods to form hybridoma cells (Goding, “Monoclonal Antibodies: Principles and Practice,” pp. 59-103 (Academic Press, 1986)). Colonies produced by individual immortalized hybridoma cells are screened to prepare antibodies having the desired properties, such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, and affinity for the antigen.

[0209] A variety of host animals can be used to generate the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies of the invention described herein. For example, Balb / c mice can be used to generate murine anti-human PSMA antibodies. A variety of techniques can be used to humanize antibodies prepared from Balb / c mice and other non-human animals to produce more human-like sequences.

[0210] Exemplary humanization techniques that involve selection of a human receptor framework are known and include CDR grafting (U.S. Patent 5,225,539), SDR grafting (U.S. Patent 6,818,749), surface reshaping (Padlan, Mol Immunol, Vol. 28, pp. 489-499, 1991), specific determining residue surface reshaping (U.S. Patent Publication 2010 / 0261620), human framework remodeling (U.S. Patent 8,748,356), or superhumanization (U.S. Patent 7,709,226). In these methods, the CDRs of the parental antibody are transferred onto a human framework, which can be selected based on its overall homology to the parental framework, based on similarity in CDR length, or canonical structural identity, or a combination thereof.

[0211] Humanized antibodies can be further optimized by the following process to improve their selectivity or affinity for a desired antigen: by introducing modified framework support residues to maintain binding affinity (back mutations), for example by using techniques such as those described in International Patent Publications WO1090 / 007861 and WO1992 / 22653, or by introducing variants at any CDR, for example, to improve the affinity of the antibody.

[0212] Transgenic animals (such as mice or rats) carrying human immunoglobulin (Ig) loci in their genomes can be used to generate human antibodies against target proteins, as described, for example, in U.S. Patent 6,150,584, International Patent Publication WO99 / 45962, International Patent Publication WO2002 / 066630, WO2002 / 43478, WO2002 / 043478, and WO1990 / 04036; Lonberg et al., 1994, Nature, Vol. 368, pp. 856-859; Green et al., 1994, Nature Genet. 7:13-21; Green & Jakobovits, 1998, Exp. Med. Vol. 188, pp. 483-495; Lonberg and Huszar, 1995, Int Rev Immunol, Vol. 13, pp. 65-93; Bruggemann et al., 1991, Eur J Immunol, Vol. 21, pp. 1323-1326; Fishwild et al., 1996, Nat Biotechnol, Vol. 14, pp. 845-851; Mendez et al., 1997, Nat Genet, Vol. 15, pp. 146-156; Green, 1999, J Immunol Methods, Vol. 231, pp. 11-23; Yang et al., 1999, Cancer Res, Vol. 59, pp. 1236-1243; Brüggemann and Taussig, 1997, Curr Opin Biotechnol, Vol. 8, pp. 455-458. The endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one intact or partial human immunoglobulin locus can be inserted into the animal's genome by homologous or non-homologous recombination using transchromosomes or minigenes. Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www_ablexis_com) can be invited to use the above techniques to provide human antibodies against selected antigens.

[0213] The human antibodies can be selected from phage display libraries, in which the phages are engineered to express human immunoglobulins or parts thereof, such as Fab, single-chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., 2000, J Mol Biol, Vol. 296, pp. 57 - 86; Krebs et al., 2001, J Immunol Meth, Vol. 254, pp. 67 - 84; Vaughan et al., 1996, Nature Biotechnology, Vol. 14, pp. 309 - 314; Sheets et al., 1998, PITAS (USA), Vol. 95, pp. 6157 - 6162; Hoogenboom and Winter, 1991, J Mol Biol, Vol. 227, p. 381; Marks et al., 1991, J Mol Biol, Vol. 222, p. 581). The antibodies of the present invention can be isolated, for example, from a phage display library expressing the heavy and light chain variable regions of the antibody as a fusion protein using the phage pIX coat protein, as described in Shi et al., 2010, J Mol Biol, Vol. 397, pp. 385 - 396 and International Patent Publication WO09 / 085462. Phages that bind to human and / or cynomolgus macaque PSMA, CD33, IL1RAP, TMEFF2, or CD3 can be screened from the library, and the obtained positive clones can be further characterized. Fab can be isolated from the clone lysates and expressed as full-length IgG. Such phage display methods for isolating human antibodies are described, for example, in U.S. Patents 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

[0214] The preparation of immunogenic antigens and the production of monoclonal antibodies can be carried out using any suitable technique such as recombinant protein production. The immunogenic antigen can be administered to an animal in the form of a purified protein or protein mixture (including whole cells or cell extracts or tissue extracts), or the antigen can be formed de novo in the animal from a nucleic acid encoding the antigen or a part thereof.

[0215] Generation and use of bispecific and multispecific CD3 antibodies

[0216] The present invention provides bispecific and multispecific antibodies, the bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. The second antigen can be a tumor-associated antigen (TAA) or an antigen on pathogenic cells.

[0217] Exemplary anti-CD3 antibodies that can be used to engineer bispecific and multispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen include CD3 antibodies that comprise the V H / V L sequences and heavy chain / light chain CDRs shown in Tables 7A and 7B, and engineered variants thereof described in Tables 10 and 11 and the accompanying text. For example, the CDRs and / or VH / VL domains of the CD3 antibodies CD3B312, CD3B313, CD3B314, CD3B315, CD3B316, CD3B317, CD3B337, CD3B373, CD3B376, CD3B389, CD3B450, and CD3B467 described herein can be used to generate bispecific and multispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen.

[0218] The anti-CD3 antibody CDRs and / or VH / VL domains described herein can be incorporated into bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen.

[0219] The anti-CD3 antibody CDRs and / or VH / VL domains described herein can be incorporated into bispecific antibodies comprising the PSMA-binding VH / VL domains described herein and in Table 23. The anti-CD3 antibodies and / or VH / VL domains described herein can be incorporated into bispecific antibodies comprising the IL1RAP-binding VH / VL domains described herein and in Table 30. The anti-CD3 antibody CDRs and / or VH / VL domains described herein can be incorporated into bispecific antibodies comprising the CD33-binding VH / VL domains described herein and in Table 38. For example, the VH / VL domains of the PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365 can be used to generate bispecific PSMA×CD3 antibodies.

[0220] Exemplary TAAs are PSMA, CD33, TMEFF2, and IL1RAP. Exemplary bispecific PSMA×CD3, CD33×CD3, TMEFF2×CD3, and IL1RAP×CD3 antibodies provided herein have a first domain that specifically binds CD3 and a second domain that specifically binds PSMA, CD33, TMEFF2, or IL1RAP. Exemplary anti-PSMA antibodies that can be used to engineer bispecific PSMA×CD3 molecules are those described herein and can comprise heavy and light chain sequences, including but not limited to the heavy and light chain sequences listed in Table 23. Exemplary anti-IL1RAP antibodies that can be used to engineer bispecific IL1RAP×CD3 molecules are those described herein and can comprise heavy and light chain variable region sequences, including but not limited to the heavy and light chain variable region sequences provided in Table 35. Exemplary anti-CD33 antibodies that can be used to engineer bispecific CD33×CD3 molecules are those described herein and can comprise heavy and light chain variable region sequences, including but not limited to the heavy and light chain variable region sequences provided in Table 43. Exemplary anti-TMEFF2 antibodies that can be used to engineer bispecific TMEFF2×CD3 molecules are those described herein and can comprise heavy and light chain variable region sequences, including but not limited to the heavy and light chain variable region sequences provided in Tables 59 and 66-68.

[0221] The resulting bispecific antibodies can be tested for binding to CD3 and / or the second antigen and / or their desired functional characteristics, such as T cell-mediated killing of cells expressing the second antigen.

[0222] Bispecific antibodies provided herein include antibodies having a full-length antibody structure.

[0223] A "Fab arm" or "half molecule" refers to one heavy chain-light chain pair that specifically binds an antigen.

[0224] Full-length bispecific antibodies as described herein can be generated, for example, by Fab-arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies by introducing replacements at the heavy chain CH3 junctions in each half-molecule to facilitate the formation of heterodimers of two antibody half-molecules with different specificities in an in vitro cell-free environment or using co-expression. The Fab-arm exchange reaction is the result of disulfide bond isomerization and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parental monospecific antibodies are reduced. The resulting free cysteines of one of the parental monospecific antibodies form inter-heavy chain disulfide bonds with cysteine residues of the second parental monospecific antibody molecule, while the CH3 domains of the parental antibodies are released and reformed by dissociation-association. The CH3 domains of the Fab arms can be engineered to facilitate heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each of which binds a different epitope, i.e., an epitope on CD3 and an epitope on a second antigen.

[0225] "Homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. "Homodimer" refers to an antibody having two heavy chains containing the same CH3 amino acid sequence.

[0226] "Heterodimerization" refers to the interaction of two heavy chains having different CH3 amino acid sequences. "Heterodimer" refers to an antibody having two heavy chains containing different CH3 amino acid sequences.

[0227] In some embodiments, bispecific antibodies include designs such as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs (Genentech), CrossMAbs (Roche), and electrostatically paired ligands (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), Strand Exchange Engineered Domain bodies (SEEDbody) (EMD Serono), Biclonic (Merus), and technologies (Genmab A / S).

[0228] The Triomab quadroma technology can be used to generate full-length bispecific antibodies that incorporate the VH and VL of the anti-CD3 antibody of the present invention. The Triomab technology facilitates Fab-arm exchange between two parental chimeric antibodies, one parental mAb having IgG2a and the second parental mAb having a rat IgG2b constant region, thereby generating a chimeric bispecific antibody.

[0229] The "button" strategy (see, for example, International Publication WO 2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions, thereby promoting heterodimer formation. An amino acid with a small side chain (button) is introduced into the heavy chain of an antibody that specifically binds a first antigen, and an amino acid with a large side chain (button) is introduced into the heavy chain of an antibody that specifically binds a second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "button" and the heavy chain with the "button". Exemplary CH3 substitution pairs that form buttons and buttons (represented as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0230] The CrossMAb technology can be used to generate full-length bispecific antibodies. In addition to using the "button" strategy to promote Fab wall exchange, the CrossMAb has an exchanged CH1 domain and CL domain in one of the half-arms to ensure correct light chain pairing of the resulting bispecific antibody (see, for example, US Patent 8,242,247).

[0231] Other exchange strategies can be used to generate full-length bispecific antibodies as follows: in one or both arms of the bispecific antibody, exchange variable domains or constant domains, or both domains, between the heavy chain and the light chain or within the heavy chain. These exchanges include, for example, VH-CH1 with VL-CL, VH with VL, CH3 with CL, and CH3 with CH1, as described in International Patent Publications WO2009 / 080254, WO2009 / 080251, WO2009 / 018386, and WO2009 / 080252.

[0232] Other strategies can also be used, such as promoting heavy chain heterodimerization using electrostatic interactions by replacing positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in U.S. Patent Publication US2010 / 0015133; U.S. Patent Publication US2009 / 0182127; U.S. Patent Publication US2010 / 028637 or U.S. Patent Publication US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (represented as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W, as described in U.S. Patent Publication US2012 / 0149876 or U.S. Patent Publication US2013 / 0195849.

[0233] The LUZ-Y technology can be used to generate bispecific antibodies. In this technology, a leucine zipper is added to the C-terminus of the CH3 domain to drive the assembly of heterodimers from parental mAbs, which are removed by post-purification, as described in Wranik et al., 2012, J Biol Chem, Vol. 287, No. 52, pp. 42221-42229.

[0234] The SEEDbody technology can be used to generate bispecific antibodies. SEEDbodies have selected IgG residues replaced with IgA residues in their constant domains to promote heterodimerization, as described in U.S. Patent US20070287170.

[0235] According to the methods described in international patent publication WO2011 / 131746, the bispecific antibodies described herein can be produced in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parental monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization. In these methods, a first monospecific bivalent antibody (i.e., an antibody that specifically binds a second antigen; e.g., an anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibody) and a second monospecific bivalent antibody (e.g., an anti-CD3 antibody) are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of the cysteines in the hinge region; thereby generating a bispecific antibody by Fab-arm exchange. The incubation conditions are restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. For example, the following conditions can be used: incubation for at least 90 minutes at a temperature of at least 20 °C, at a pH of 5-8 such as pH 7.0 or pH 7.4, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol.

[0236] In some embodiments described herein, a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen comprises at least one substitution in the antibody CH3 constant domain.

[0237] In some embodiments described herein, at least one substitution in the antibody CH3 constant domain is a K409R, F405L, or F405L and R409K substitution, where the residues are numbered according to the EU index.

[0238] Antibody domains and position numbering are well known. "Asymmetric" refers to different substitutions within the two CH3 domains in the two independent heavy chains of the antibody. The IgG1 CH3 region is typically composed of residues 341-446 on IgG1 (residues are numbered according to the EU index).

[0239] In some embodiments described herein, the bispecific antibody is of the IgG1 isotype and comprises an F405L substitution in the first heavy chain (HC1) of the antibody and a K409R substitution in the second heavy chain (HC2) of the antibody when compared to wild-type IgG1.

[0240] In some embodiments described herein, the bispecific antibody is of the IgG1 isotype and, when compared to wild-type IgG1, contains a K409R substitution in the first heavy chain (HC1) of the antibody and an F405L substitution in the second heavy chain (HC2) of the antibody.

[0241] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and, when compared to wild-type IgG4, contains an S228P substitution in HC1 and S228P, F405L, and R409K substitutions in HC2.

[0242] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and, when compared to wild-type IgG4, contains S228P, F405L, and R409K substitutions in HC1 and an S228P substitution in HC2.

[0243] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and, when compared to wild-type IgG4, contains S228P, F234A, and L235A substitutions in HC1 and S228P, F234A, L235A, F405L, and R409K substitutions in HC2.

[0244] In some embodiments described herein, the bispecific antibody is of the IgG4 isotype and, when compared to wild-type IgG4, contains S228P, F234A, L235A, F405L, and R409K substitutions in HC1 and S228P, F234A, and L235A substitutions in HC2.

[0245] In some embodiments described herein, when residues are numbered according to the EU index, the bispecific antibody of the present invention contains at least one, two, three, four, five, six, seven, or eight asymmetric substitutions at residue positions 350, 366, 368, 370, 399, 405, 407, or 409 in HC1 and HC2.

[0246] In some embodiments described herein, when residues are numbered according to the EU index, the bispecific antibody of the present invention contains at least one, two, three, or four asymmetric substitutions at residue positions 350, 370, 405, or 409 in HC1 and HC2.

[0247] In some embodiments described herein, when residues are numbered according to the EU index, the bispecific antibody of the present invention contains at least one asymmetric substitution at residue positions 405 or 409 in HC1 and HC2.

[0248] Standard methods are typically used to perform replacements at the DNA level up to the molecular level (such as the constant domain of an antibody).

[0249] The antibodies of the present invention can be engineered into various well-known antibody forms.

[0250] In some embodiments, the bispecific antibodies of the present invention are crossbodies.

[0251] In some embodiments, the bispecific antibodies of the present invention include recombinant IgG-like dual-targeting molecules, where each side of the molecule contains at least two Fab fragments or portions of Fab fragments of different antibodies; IgG fusion molecules, where a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; Fc fusion molecules, where a single-chain Fv molecule or a stable diabody antibody is fused to a heavy-chain constant domain, Fc region, or a portion thereof; Fab fusion molecules, where different Fab fragments are fused together; single-chain Fv (ScFv)- and diabody-based heavy-chain antibodies (such as domain antibodies, nanobodies), where different single-chain Fv molecules or different diabody antibodies or different heavy-chain antibodies (such as domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0252] In some embodiments, the recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK / Domantis), diabody (Genentech), and mAb2 (F-Star).

[0253] In some embodiments, the IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

[0254] In some embodiments, the Fc fusion molecules include ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), and bifunctional affinity retargeting technology (Fc-DART) (MacroGenics).

[0255] In some embodiments, Fab-fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol), and Fab-Fv (UCB-Celltech). ScFv-based and diabody-based domain antibodies include bispecific T cell engagers (BITE) (Micromet), tandem diabodies (Tandab) (Affimed), dual-affinity retargeting molecules (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), and dual-targeting heavy chain domain antibodies only. Various forms of bispecific antibodies have been described, for example, in Chames and Baty, 2009, Curr Opin Drug Disc Dev, Vol. 12, p. 276 and Nunez-Prado et al., 2015, Drug Discovery Today, Vol. 20, No. 5, pp. 588-594.

[0256] Table 2 summarizes the exemplary monoclonal antibodies described herein that can be used to generate the bispecific antibodies of the present invention.

[0257] Table 2. Exemplary monoclonal antibodies that can be used to generate the bispecific antibodies of the invention

[0258]

[0259] Exemplary anti-TMEFF2 antibodies that can be used to engineer bispecific TMEFF2×CD3 molecules are those described herein and may comprise heavy and light chain variable region sequences, including but not limited to the heavy and light chain variable region sequences provided in Tables 59 and 66-68.

[0260] In some embodiments, the anti-CD3 antibody is a multispecific antibody, such as a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen. In some embodiments, the second antigen or target antigen is a cell surface antigen expressed on target cells other than immune effector cells. In some embodiments, the second antigen is a TAA. Exemplary TAAs are PSMA, CD33, TMEFF2, and IL1RAP.

[0261] The present invention provides a bispecific antibody that comprises a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen.

[0262] In some embodiments, the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 690, respectively. In some embodiments, the first domain comprises VH and VL that are SEQ ID NO:652 and 661, respectively. In some embodiments, the first domain comprises HC and LC that are SEQ ID NO:640 and 676, respectively. In some embodiments, the first domain comprises VH and VL that are SEQ ID NO:657 and 678, respectively. In some embodiments, the first domain comprises HC and LC that are SEQ ID NO:675 and 677, respectively.

[0263] In some embodiments, the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 773, 673, and 690, respectively. In some embodiments, the first domain comprises VH and VL that are SEQ ID NO:657 and 678, respectively. In some embodiments, the first domain comprises HC and LC that are SEQ ID NO:675 and 677, respectively.

[0264] The present invention also provides a bispecific antibody for use in therapy, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention.

[0265] The present invention also provides a bispecific antibody for use in treating a proliferative disorder, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention.

[0266] The present invention also provides a bispecific antibody for use in treating cancer, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention.

[0267] The present invention also provides a bispecific antibody for use in treating an autoimmune disease, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention.

[0268] The present invention also provides a bispecific antibody for use in the preparation of a medicament for treating cancer, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention.

[0269] The present invention also provides a bispecific antibody for use in the preparation of a medicament for treating an autoimmune disease, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention.

[0270] Another aspect of the present invention is a method of treating a cell proliferative disease or an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the anti-CD3 antibody of the present invention. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention is administered to the subject at a dose of about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention is administered to the subject at a dose of about 1 mg / kg. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention is administered subcutaneously. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention is administered intravenously.

[0271] In any of the foregoing uses or methods, the proliferative disease is cancer. In some embodiments, the cancer is selected from esophageal cancer, gastric cancer, small intestine cancer, large intestine cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenström macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassified splenic diffuse red pulp small B-cell lymphoma, variant hairy cell leukemia, Waldenström macroglobulinemia, heavy chain disease, plasmacytoma, solitary plasmacytoma of bone, extramedullary plasmacytoma, mucosa-associated lymphoid tissue extranodal marginal zone lymphoma (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system DLBCL, primary cutaneous DLBCL - leg type, elderly EBV-positive DLBCL, chronic inflammation-associated DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma caused by HHV8-related multicentric Castleman disease, primary effusion lymphoma: an unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and an unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma, classical Hodgkin lymphoma, and light chain amyloidosis.

[0272] In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is adenocarcinoma, such as metastatic adenocarcinoma (e.g., colorectal adenocarcinoma, gastric adenocarcinoma, or pancreatic adenocarcinoma).

[0273] In any of the foregoing uses or methods, the autoimmune disease may be selected from rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Raynaud's syndrome, Sjogren's syndrome, glomerulonephritis, neuromyelitis optica (NMO), and IgG neuropathy.

[0274] In another aspect, the invention features a kit that includes: (a) a composition comprising any one of the foregoing anti-CD3 antibodies or bispecific antibodies, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention, and (b) package insert instructions that include instructions for administering the composition to a subject to treat or delay the progression of a cell proliferative disease. The term "package insert instructions" is used to refer to the instructions that are typically included in the commercial packaging of a therapeutic product and that contain information regarding indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings relating to the use of such therapeutic product.

[0275] In any of the foregoing uses or methods, the subject can be a human.

[0276] Polynucleotides, vectors, and host cells

[0277] Also disclosed is an isolated polynucleotide encoding the anti-CD3 antibody of the invention or the bispecific antibody of the invention, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen. Isolated polynucleotides capable of encoding the variable domains provided herein can be included on the same or different vectors to produce the antibodies or antigen-binding fragments of the invention.

[0278] In some embodiments, the polynucleotide of the invention includes a polynucleotide encoding a leader sequence. Any leader sequence known in the art can be employed. The polynucleotide encoding the leader sequence can include a restriction endonuclease cleavage site or a translation initiation site.

[0279] Also provided is a vector comprising the polynucleotide of the invention. The vector can be an expression vector. The expression vector can contain one or more additional sequences such as, but not limited to, regulatory sequences (e.g., promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0280] Recombinant expression vectors within the scope of this specification include synthetic or cDNA-derived nucleic acid fragments that encode at least one recombinant protein operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. Expression vectors, particularly mammalian expression vectors, may also contain one or more non-transcribed elements, such as an origin of replication, suitable promoters and enhancers linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' untranslated sequences (such as essential ribosome binding sites), polyadenylation sites, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication conferring the ability to replicate in the host may also be incorporated.

[0281] The transcriptional and translational control sequences in expression vectors for transforming vertebrate cells may be provided from a viral source. Exemplary vectors are as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0282] In some embodiments, the antibody-encoding sequence or antigen-binding fragment-encoding sequence is placed under the control of a strong constitutive promoter (such as the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin, human myosin, human hemoglobin, human muscle creatine, etc.). In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) of Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. In one embodiment, the PSMA-specific antibody or its antigen-binding fragment-encoding sequence is placed under the control of an inducible promoter (such as the metallothionein promoter, the tetracycline-inducible promoter, the doxycycline-inducible promoter, a promoter containing one or more interferon-stimulated response elements (ISREs) (such as protein kinase R 2',5'-oligoadenylate synthetase, Mx gene, ADAR1, etc.)).

[0283] The vectors described herein may contain one or more internal ribosome entry sites (IRESs). Inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing the expression of some proteins. In some embodiments, the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the nucleic acid sequences described above. The vector components may be joined continuously, or arranged in a manner that provides optimal spacing for the expression of the gene product (i.e., by introducing "spacer" nucleotides between the ORFs), or positioned in another way. Regulatory elements such as the IRES motif may also be arranged to provide optimal spacing for expression.

[0284] The vector may contain selectable markers well known in the art. Selectable markers include positive and negative selectable markers, e.g., antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene), glutamate synthase gene, HSV-TK, an HSV-TK derivative for ganciclovir selection or a bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence encoding the selectable marker or cloning site may be upstream or downstream of the nucleic acid sequence encoding the polypeptide of interest or the cloning site.

[0285] The vectors described herein can be used to transform various cells with the gene encoding the antibody or antigen-binding fragment. For example, the vector can be used to generate cells that produce anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2 or anti-IL1RAP antibodies or antigen-binding fragments. Accordingly, the present invention also provides a host cell comprising the vector of the present invention.

[0286] A variety of techniques are known in the art for introducing foreign genes into cells, and for the purposes of practicing the methods described herein, these techniques can be used to construct recombinant cells according to the various embodiments described and illustrated herein. The techniques used should result in the stable transfer of the heterologous gene sequence into the host cell such that the heterologous gene sequence is heritable and expressible by the progeny of the cell, and thus the necessary developmental and physiological functions of the recipient cell are not disrupted. Techniques that can be used include, but are not limited to, chromosome transfer (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome vectors), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses), etc. (described in Cline, 29 Pharmac. Ther. pp. 69-92, 1985). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.

[0287] Cells suitable for use in expressing the antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent or human origin, such as, but not limited to, NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and BHK cell lines, etc. In addition, hybridoma cells can be used to accomplish the expression of antibodies. Methods for generating hybridomas are well established in the art.

[0288] Cells transformed with the expression vectors of the present invention can be selected or screened for recombinant expression of the antibodies or antigen-binding fragments of the present invention. Recombinant positive cells are amplified and screened, and subclones are screened that exhibit desired phenotypes, such as high-level expression, enhanced growth characteristics, or the ability to produce proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modification. These phenotypes may be due to the inherent properties of a given subclone or due to mutations. Mutations can be achieved by using chemicals, UV wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or combinations of these methods.

[0289] Pharmaceutical compositions / Administration

[0290] The present invention also provides a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibody of the present invention can be prepared as a pharmaceutical composition, which contains an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient or vehicle with which the antibody of the present invention is administered. Such vehicles can be liquids, such as water and oils, including those derived from petroleum, animals, plants or synthetic ones, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and usually free of particulate matter. They can be sterilized by conventional well-known sterilization techniques (such as filtration). The composition can contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH regulators and buffers, stabilizers, thickeners, lubricants and coloring agents, etc. The concentration of the antibody of the present invention in such pharmaceutical preparations can vary from less than about 0.5% by weight, usually to at least about 1% up to 15 or 20%, and can be selected according to the specific mode of administration chosen, mainly based on the required dose, fluid volume, viscosity, etc. Suitable vehicles and formulations including other human proteins (such as human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Troy, D.B., Lipincott Williams and Wilkins, Philadelphia, PA, 2006, Part 5, Pharmaceutical Manufacturing, pp. 691 - 1092 (see especially pp. 958 - 989).

[0291] The mode of administration for the therapeutic use of the antibody of the present invention can be any suitable route for delivering the antibody to a host, such as parenteral administration, for example intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using preparations in the form of tablets, capsules, solutions, powders, gels, granules; and contained in syringes, implant devices, osmotic pumps, cartridges, micropumps; or other ways well-known in the art understood by those skilled in the art. Site-specific administration can be achieved, for example, by intratumoral, parenteral, intratracheal, intra-abdominal, intracapsular, intrachondral, intracavitary, intracoelomic, intracerebellar, intraventricular, intracolonic, endocervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, pericardial, intraperitoneal, intrathoracic, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, oral, sublingual, intranasal or transdermal delivery.

[0292] The antibodies of the present invention can be administered to a subject by any suitable route, such as parenterally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally. The i.v. infusion can be given over, for example, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes or 240 minutes or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.

[0293] The dose administered to the subject is sufficient to alleviate or at least partially arrest the disease being treated ("therapeutically effective amount"), and can sometimes be from 0.005 mg / kg to about 100 mg / kg, such as about 0.05 mg / kg to about 30 mg / kg, or about 5 mg / kg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg or about 24 mg / kg, or for example about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg, but can even be higher, such as about 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg.

[0294] Fixed unit doses can also be given, such as 50 mg, 100 mg, 200 mg, 500 mg or 1000 mg, or the dose can be based on the patient's surface area, such as 500 mg / m2, 400 mg / m2, 300 mg / m2, 250 mg / m2, 200 mg / m2 or 100 mg / m2. Usually between 1 and 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) can be administered to treat the patient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses can be given.

[0295] The antibody of the present invention can be repeatedly administered after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. The treatment process can also be repeated, in the same manner as chronic administration. The repeated administration can be at the same dose or different doses. For example, the antibody of the present invention described herein can be administered by intravenous infusion at 8 mg / kg or at 16 mg / kg at weekly intervals for 8 weeks, then at 8 mg / kg or at 16 mg / kg every two weeks for another 16 weeks, and then at 8 mg / kg or at 16 mg / kg every four weeks.

[0296] For example, the antibody in the method described herein can be provided as a daily dose in an amount of about 0.1 - 100 mg / kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg / day, at at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 after the start of treatment, or alternatively, at at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using a single dose or divided doses every 24, 12, 8, 6, 4 or 2 hours or any combination thereof.

[0297] The antibody in the method described herein can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression and / or reduce the risk of recurrence after cancer remission.

[0298] The antibody provided herein can be lyophilized under reduced pressure for storage and reconstituted in a suitable vehicle before use. This technique has been proven effective for conventional protein preparations and well-known lyophilization and reconstitution techniques can be employed.

[0299] Methods of detecting CD3, PSMA, CD33, IL1RAP, TMEFF2, or a target antigen and CD3

[0300] The present disclosure provides methods for detecting CD3, PSMA, CD33, TMEFF2, or IL1RAP in a sample, the methods comprising obtaining a sample, contacting the sample with an anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibody of the present disclosure, and detecting the antibody that binds to CD3, PSMA, CD33, TMEFF2, or IL1RAP in the sample.

[0301] Also provided are methods for detecting CD3 and a second antigen (e.g., PSMA, CD33, TMEFF2, or IL1RAP) in a sample, the methods comprising obtaining a sample, contacting the sample with a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds the second antigen, and detecting the antibody that binds to CD3 and the second antigen in the sample.

[0302] In some embodiments described herein, the sample can be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspirate tissue), histological preparations, and the like.

[0303] The antibodies of the present disclosure can be detected using known methods. Exemplary methods include directly labeling the antibody with a fluorescent or chemiluminescent label, or a radiolabel, or attaching a moiety that is amenable to detection (such as biotin, an enzyme, or an epitope tag) to the antibody of the present disclosure. Exemplary labels and moieties are ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorescein dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, and dyes.

[0304] The antibodies provided herein can be used in a variety of assays to detect CD3, PSMA, CD33, IL1RAP, TMEFF2, or CD3 and a second antigen in a sample. Exemplary assays are Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA assay.

[0305] Antibody kits

[0306] The present invention also provides a kit comprising one or more of an anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2 or anti-IL1RAP antibody or bispecific antibody, wherein the bispecific antibody comprises a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention or an antigen-binding fragment thereof. The kit can be used to implement a method using an anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2 or anti-IL1RAP antibody or a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the present invention, or other methods known to those skilled in the art. In some embodiments, the kit may include the antibodies or antigen-binding fragments described herein and reagents for use in detecting the presence of CD3 or a second antigen (such as PSMA, CD33, IL1RAP, TMEFF2) in a biological sample. Thus, the kit may include one or more of the following: an antibody or an antigen-binding fragment thereof described herein, and a container for accommodating the antibody or fragment when not in use, instructions for use of the antibody or fragment, an antibody or fragment attached to a solid-phase support, and / or an antibody or fragment in a detectable-labeled form, as described herein.

[0307] The present invention also provides a kit comprising an antibody that specifically binds PSMA described herein. The present invention also provides a kit comprising an antibody that specifically binds CD33 described herein. The present invention also provides a kit comprising an antibody that specifically binds IL1RAP described herein. The present invention also provides a kit comprising an antibody that specifically binds TMEFF2 described herein.

[0308] The kit can be used for therapeutic purposes and as a diagnostic kit.

[0309] The kit can be used to detect the presence of CD3, PSMA, CD33, IL1RAP, TMEFF2, and / or a second antigen in a biological sample.

[0310] In some embodiments, the kit comprises the antibody of the present invention described herein and a reagent for detecting the antibody. The kit may comprise one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or reagents that can be used to chelate or otherwise conjugate the antibody to a label or therapeutic agent, or a radiation protection composition; a device or other material for preparing the antibody for administration; a pharmaceutically acceptable carrier; and a device or other material for administering to a subject.

[0311] In some embodiments, the kit comprises the antibody of the present invention in a container and instructions for use of the kit.

[0312] In some embodiments, the antibody in the kit is labeled.

[0313] CD3-specific antibodies

[0314] This document describes isolated anti-CD3 antibodies. The anti-CD3 antibodies of the present invention bind to human CD3 and optionally cynomolgus monkey CD3. In some embodiments, the anti-CD3 antibodies of the present invention and their fragments bind to human CD3 and cynomolgus monkey CD3 with an affinity within 5-fold of each other. In other words, the difference in antibody binding is less than 5-fold. In such cases, the anti-CD3 antibodies of the present invention can be used both for preclinical evaluation of the safety, activity, and / or pharmacokinetic characteristics of CD3 in primates and as a drug for humans. In other words, the same CD3-specific molecule can be used for preclinical animal studies as well as for human clinical studies. Compared with species-specific surrogate molecules, this human / cynomolgus monkey cross-reactivity results in highly comparable results in animal studies and a greatly increased predictive power. In some embodiments, the anti-CD3 antibodies of the present invention and their fragments bind to an epitope formed by the CD3e / d subunits. The CD3-specific antibody can be human, humanized, or chimeric. Also illustrated herein are human antibodies generated in OmniRat (Open Monocmatic Technologies (“OMT”), Palo Alto, California, USA, omniab.com).

[0315] In some embodiments described herein, the anti-CD3 antibody or its fragment has one, two, three, four, or five of the following characteristics:

[0316] a) binds to human and cynomolgus macaque CD3+ T lymphocytes with a calculated EC50 of 20 nM or less and binds to HEK cells expressing cynomolgus monkey CD3 with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding to CD3+ T lymphocytes and binding to HEK cells expressing cynomolgus monkey CD3 is less than 5-fold, and wherein the calculated EC50 is measured by flow cytometry in a whole cell binding assay at 0 °C;

[0317] b) binds to recombinant CD3d D from humans with an equilibrium dissociation constant (K

[0318] (SEQ ID NO: 691), or binds to recombinant CD3e from humans (SEQ ID NO:

[0319] 636), or binds to recombinant CD3e from cynomolgus monkeys (SEQ ID NO: 693), wherein K D is measured using a Proteon surface plasmon resonance assay on a ProteOn XPR36 system at +25 °C;

[0320] c) showing no methionine or tryptophan oxidation, or showing no asparagine deamidation, or showing no asparagine isomerization, as detected by peptide mapping;

[0321] d) binding to residues 1 - 6 of CD3e, as determined by X - ray crystallography; or

[0322] e) activating T cells or inducing CD69 expression to a level similar to cOKT3 or SP34 - 2, as determined by fluorescence - activated cell sorting assay.

[0323] The anti - CD3 antibodies and fragments thereof of the present invention have an in vitro binding affinity (K d ) of about 5 nM to about 1000 nM, preferably about 5 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, and about 900 nM to about 1000 nM, more preferably about 5 nM to about 300 nM, for human T cells expressing human CD3, as determined by flow cytometry.

[0324] In some aspects, the anti - CD3 antibodies and fragments thereof of the present invention are bivalent antibodies that have an in vitro binding affinity (K d ) of about 5 nM to about 1000 nM, preferably about 5 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, and about 900 nM to about 1000 nM, more preferably about 5 nM to about 300 nM, most preferably about 100 nM, for human T cells expressing human CD3, as determined by flow cytometry.

[0325] In some aspects, the anti-CD3 antibodies and fragments thereof of the present invention are monovalent constructs that have an in vitro binding affinity (K d ) for human T cells expressing human CD3 of from about 5 nM to about 1000 nM, preferably from about 5 nM to about 50 nM, from about 50 nM to about 100 nM, from about 100 nM to about 200 nM, from about 200 nM to about 300 nM, from about 300 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, and from about 900 nM to about 1000 nM, more preferably from about 100 nM to about 250 nM, and most preferably about 250 nM, as determined by flow cytometry.

[0326] In one aspect, the anti-CD3 antibodies and fragments thereof described herein compete for binding to CD3 with the commercially available CD3 antibody SP34-2 (BD Biosciences 551916), as determined by a competitive binding assay using Alexa Fluor 488-conjugated SP34-2 antibody to primary human T cells, which competitive binding assay is measured using flow cytometry.

[0327] In one aspect, the anti-CD3 antibodies and fragments thereof do not exhibit post-translational modifications, including no oxidation, no deamidation, and no aspartic acid isomerization, as determined by peptide mapping.

[0328] In one aspect, the anti-CD3 antibodies and fragments thereof are effective in activating T cells and inducing CD69 expression to a level similar to that of SP34-2 in human and cynomolgus monkey T cells and cOKT3 in human T cells, as determined by T cell-based assays using flow cytometry.

[0329] In one aspect, the anti-CD3 antibodies and fragments thereof described herein have a total unfolding enthalpy of about 400 kcal / mol or greater, about 410 kcal / mol or greater, about 420 kcal / mol or greater, about 430 kcal / mol or greater, about 440 kcal / mol or greater, about 45 kcal / mol or greater, about 460 kcal / mol or greater, about 470 kcal / mol or greater, about 480 kcal / mol or greater, about 490 kcal / mol or greater, about 500 kcal / mol or greater, about 510 kcal / mol or greater, about 520 kcal / mol or greater, about 530 kcal / mol or greater, about 540 kcal / mol or greater or about 550 kcal / mol or greater. In certain aspects, the anti-CD3 antibodies and fragments thereof of the invention have a total unfolding enthalpy of about 418 kcal / mol, 545 kcal / mol, about 402 kcal / mol or about 406 kcal / mol, and the anti-CD3 antibodies are CD3B376 (IgG4 PAA) molecule, CD3B450 (IgG4 PAA) molecule, CD3B389 (IgG1σ) molecule and CD3B467 (IgG1σ) molecule, respectively.

[0330] Exemplary such antibodies include CD3B311, CD3B312, CD3B313, CD3B314, CD3B315, CD3B316, CD3B317, CD3B334, CD3B376, CD3B389, CD3B450 and CD3B467, and CD3B376 and CD3B450 engineered to be in monovalent form.

[0331] The anti-CD3 antibodies or antigen-binding fragments of the invention may exist in a variety of forms, but will include one or more of the antibody variable domain fragments or CDRs shown in Table 7A and their engineered variants, e.g., those shown or described in Tables 9 and 10 and their accompanying descriptions.

[0332] The present invention also provides anti-CD3 antibodies or antigen-binding fragments thereof, which comprise a heavy chain containing HCDR1, HCDR2, and HCDR3 of any one of the antibodies described in Table 7B. The present invention also provides anti-CD3 antibodies or antigen-binding fragments thereof, which comprise a heavy chain containing HCDR1, HCDR2, and HCDR3 of any one of the antibodies described in Table 7B and a light chain containing LCDR1, LCDR2, and LCDR3 of any one of the antibodies described in Table 7B. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention competes with an antibody or antigen-binding fragment thereof comprising a heavy chain containing HCDR1, HCDR2, and HCDR3 of any one of the antibodies described in Table 7B and a light chain containing LCDR1, LCDR2, and LCDR3 of any one of the antibodies described in Table 7B for binding to CD3.

[0333] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, and HCDR3 contained within the heavy chain variable region (VH) of SEQ ID NO: 651, 652, 653, 654, 655, 687, or 656, wherein HCDR1, HCDR2, and HCDR3 are defined by Chothia, Kabat, or IMGT.

[0334] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises LCDR1, LCDR2, and LCDR3 contained within the light chain variable region (VH) of SEQ ID NO: 658, 659, 694, 660, 688, or 661, wherein LCDR1, LCDR2, and LCDR3 are defined by Chothia, Kabat, or IMGT.

[0335] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises:

[0336] HCDR1 of SEQ ID NO: 662, 665, or 666;

[0337] HCDR2 of SEQ ID NO: 663, 689, or 695; and

[0338] HCDR3 of SEQ ID NO: 664;

[0339] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises:

[0340] LCDR1 of SEQ ID NO: 773, 710, 674, or 671;

[0341] LCDR2 of SEQ ID NO: 669 or 673; and

[0342] The LCDR3 of SEQ ID NO: 670.

[0343] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises:

[0344] The HCDR1 of SEQ ID NO: 662, 665 or 666;

[0345] The HCDR2 of SEQ ID NO: 663, 689 or 695;

[0346] The HCDR3 of SEQ ID NO: 664;

[0347] The LCDR1 of SEQ ID NO: 773, 710, 674 or 671;

[0348] The LCDR2 of SEQ ID NO: 669 or 673; and

[0349] The LCDR3 of SEQ ID NO: 670.

[0350] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises the following HCDR1, HCDR2 and HCDR3:

[0351] SEQ ID NO: 662, 663 and 664, respectively;

[0352] SEQ ID NO: 662, 695 and 664, respectively;

[0353] SEQ ID NO: 665, 663 and 664, respectively;

[0354] SEQ ID NO: 665, 695 and 664, respectively;

[0355] SEQ ID NO: 662, 689 and 664, respectively;

[0356] SEQ ID NO: 666, 663 and 664, respectively;

[0357] SEQ ID NO: 666, 695 and 664, respectively;

[0358] SEQ ID NO: 665, 689 and 664, respectively; or

[0359] SEQ ID NO: 666, 689 and 664, respectively.

[0360] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises the following LCDR1, LCDR2, and LCDR3:

[0361] SEQ ID NO: 773, 669, and 670, respectively;

[0362] SEQ ID NO: 773, 673, and 670, respectively;

[0363] SEQ ID NO: 710, 673, and 670, respectively;

[0364] SEQ ID NO: 674, 673, and 670, respectively;

[0365] SEQ ID NO: 671, 673, and 690, respectively;

[0366] SEQ ID NO: 773, 673, and 690, respectively;

[0367] SEQ ID NO: 671, 669, and 670, respectively; or

[0368] SEQ ID NO: 776, 673, and 670, respectively.

[0369] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 773, 669, and 670, respectively.

[0370] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 773, 673, and 670, respectively.

[0371] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 671, 669, and 670, respectively.

[0372] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 671, 673, and 690, respectively.

[0373] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 695, 664, 773, 669, and 670, respectively.

[0374] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 695, 664, 773, 673, and 670, respectively.

[0375] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 695, 664, 671, 669, and 670, respectively.

[0376] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 695, 664, 671, 673, and 670, respectively.

[0377] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:665, 663, 664, 773, 669, and 670, respectively.

[0378] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:665, 663, 664, 773, 673, and 670, respectively.

[0379] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:665, 663, 664, 671, 669, and 670, respectively.

[0380] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:665, 663, 664, 671, 673, and 670, respectively.

[0381] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 665, 695, 664, 773, 669, and 670, respectively.

[0382] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 665, 695, 664, 773, 673, and 670, respectively.

[0383] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 665, 695, 664, 776, 669, and 670, respectively.

[0384] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 665, 695, 664, 776, 673, and 670, respectively.

[0385] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 666, 663, 664, 773, 669, and 670, respectively.

[0386] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 666, 663, 664, 773, 673, and 670, respectively.

[0387] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 666, 663, 664, 776, 669, and 670, respectively.

[0388] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:666, 663, 664, 671, 673, and 670, respectively.

[0389] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:666, 695, 664, 773, 669, and 670, respectively.

[0390] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:666, 695, 664, 773, 673, and 670, respectively.

[0391] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:666, 695, 664, 671, 669, and 670, respectively.

[0392] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:666, 695, 664, 671, 673, and 670, respectively.

[0393] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 689, 664, 671, 673, and 670, respectively.

[0394] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 710, 673, and 670, respectively.

[0395] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 690, respectively.

[0396] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 773, 673, and 690, respectively.

[0397] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain (HC) sequence of SEQ ID NO: 709, 640, 641, 642, 643, 675, or 644 and / or a light chain (LC) sequence of SEQ ID NO: 645, 716, 649, 676, 677, or 650. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises an HC and an LC, wherein the HC has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 709, 640, 641, 642, 643, 675, or 644, and the LC has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 645, 716, 649, 676, 677, or 650. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises an HC and an LC, wherein the HC has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 709, 640, 641, 642, 643, 675, or 644, and the LC has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 645, 716, 649, 676, 677, or 650, wherein the sequence variations do not occur in the CDR regions.

[0398] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) sequence of SEQ ID NO: 651, 652, 657, 653, 654, 655, 687 or 656 and / or a light chain variable region (VL) sequence of SEQ ID NO: 658, 659, 694, 660, 688, 678 or 661. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises a VH and a VL, the VH having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 651, 652, 657, 653, 654, 655, 687 or 656, and the VL having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 658, 659, 694, 660, 688, 678 or 661. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises a VH and a VL, the VH having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 651, 652, 657, 653, 654, 655, 687 or 656, and the VL having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 658, 659, 694, 660, 688, 678 or 661, wherein the sequence variations do not occur in the CDR regions.

[0399] PSMA-specific antibodies

[0400] The antibody and its fragment that bind to PSMA bind to the chimpanzee target antigen. In one embodiment, the antibody and its fragment bind to the human and macaque PSMA target antigens with an affinity within 5-fold of each other. In other words, the difference in antibody binding is less than 5-fold. In this case, the same antibody molecule can be used for preclinical evaluation of the safety, activity, and / or pharmacokinetic characteristics of PSMA in primates and also as a drug for humans. In other words, the same PSMA-specific molecule can be used for preclinical animal studies as well as for human clinical studies. This results in highly comparable results in animal studies and a greatly increased predictive power compared to species-specific surrogate molecules. Since the PSMA domain is cross-species specific, i.e., reactive with human and macaque antigens, the antibody or its fragment of the present invention can be used for preclinical evaluation of the safety, activity, and / or pharmacokinetic characteristics of these binding domains in primates and also in the same form as a human drug.

[0401] The present invention also provides multispecific antibodies that specifically bind to PSMA. According to the present invention, bispecific (i.e., bifunctional) antibodies can be used to engage two different therapeutic targets or perform two different functions. Such antibodies can be used, for example, to recruit immune effector cells, such as T cells or NK cells, to specific target cells. A variety of antibody fragment-based molecules are known and are under investigation, for example, for cancer treatment.

[0402] The present invention also provides PSMA×"effector antigen" bispecific antibodies. In one embodiment, the effector antigen of the PSMA×"effector antigen" bispecific antibody is CD3. It has been found in the present invention that PSMA×CD3 bispecific antibodies can be produced, and the same molecule can be used for preclinical animal testing as well as clinical studies and even for human treatment. This is due to the identification of a PSMA×CD3 bispecific antibody that binds not only to human PSMA and human CD3 respectively, but also to antigen homologues in chimpanzees and macaques. The PSMA×CD3 bispecific antibody of the present invention can be used as a therapeutic agent for various diseases, including but not limited to cancer. In view of the above, there is no longer a need to construct alternative target PSMA×CD3 bispecific antibodies for testing in phylogenetically distant (from humans) species. Therefore, the same molecule can be used for animal preclinical testing, as in clinical testing and as intended for human administration after market approval and therapeutic drug administration.

[0403] In some embodiments described herein, the isolated antibody or antibody fragment that specifically binds to PSMA has one, two, three, four, or five of the following characteristics:

[0404] a. Binds to the extracellular domain (ECD) of chimpanzee PSMA with an equilibrium dissociation constant (K D ) of less than 25 nM or less, where K D is measured using a ProteOn XPR36 system at +25°C,

[0405] b. Binds to LNCaP lymphocytes with a calculated EC50 of 20 nM or less and binds to HEK cells expressing cynomolgus macaque PSMA with a calculated EC50 of 40 nM or less, where the difference in calculated EC50 between binding to LNCaP cells and binding to HEK cells expressing cynomolgus macaque PSMA is less than 5-fold, and where the calculated EC50 is measured by flow cytometry in a whole-cell binding assay at 0°C,

[0406] c. Binds to the extracellular domain (ECD) of chimpanzee PSMA with an equilibrium dissociation constant (K D) in combination with recombinant PSMA ECD from human (SEQ ID NO:55), recombinant PSMA ECD from chimpanzee (SEQ ID NO:52), or recombinant PSMA ECD from cynomolgus macaque (SEQ ID NO:53), wherein K D Measured using a Proteon surface plasmon resonance ProteOn XPR36 system at +25 °C;

[0407] d. When paired with an anti-CD3 antibody in a bispecific antibody, shows T cell-mediated killing of LNCaP cells, C42 cells, HEK cells expressing human PSMA, or HEK cells expressing cynomolgus macaque PSMA, wherein T cell-mediated killing is measured by chromium-51 or caspase 3 / 7 activation assays, or

[0408] e. Recognizes a conformational epitope, wherein the epitope is composed of residues

[0409] I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324 - P326 of human PSMA (SEQ ID NO:51)

[0410] Exemplary such antibodies or fragments thereof are the PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365 described herein.

[0411] In some embodiments of the invention described herein, the antibody that specifically binds PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:56, 57, 58, 59, 60, and 61, respectively.

[0412] In some embodiments of the invention described herein, the antibody that specifically binds PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:62, 63, 64, 65, 60, and 66, respectively.

[0413] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:67, 68, 69, 70, 71, and 72, respectively.

[0414] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:73, 74, 75, 76, 60, and 61, respectively.

[0415] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 79, 80, 81, 82, and 83, respectively.

[0416] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:84, 85, 86, 87, 60, and 88, respectively.

[0417] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:89, 90, 91, 92, 93, and 94, respectively.

[0418] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:95, 96, 97, 65, 60, and 66, respectively.

[0419] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:84, 98, 99, 100, 82, and 101, respectively.

[0420] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:89, 90, 102, 103, 104, and 105, respectively.

[0421] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:89, 90, 106, 103, 104, and 105, respectively.

[0422] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:107, 108, 109, 76, 60, and 88, respectively.

[0423] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 1, 80, 81, 82, and 83, respectively.

[0424] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 1, 80, 81, 82, and 83, respectively.

[0425] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 1, 80, 4, 82, and 686, respectively.

[0426] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 1, 80, 81, 792, and 686, respectively.

[0427] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 2, 80, 81, 82, and 83, respectively.

[0428] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 3, 80, 81, 82, and 5, respectively.

[0429] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 3, 80, 81, 82, and 83, respectively.

[0430] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 3, 80, 4, 82, and 686, respectively.

[0431] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 3, 80, 81, 792, and 686, respectively.

[0432] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 2, 81, 81, 82, and 5, respectively.

[0433] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 2, 80, 4, 792, and 686, respectively.

[0434] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 2, 80, 4, 792, and 686, respectively.

[0435] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:78, 683, 80, 81, 792, and 686, respectively.

[0436] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA comprises a heavy chain variable region (VH) of SEQ ID NO:6, 7, 8, 110, 112, 114, 116, 118, 120, 121, 123, 125, 126, 128, 130, or 681. In some embodiments of the invention described herein, the antibody that specifically binds to PSMA comprises a light chain variable region (VL) of SEQ ID NO:9, 111, 113, 115, 117, 119, 122, 124, 127, 129, 131, or 682.

[0437] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA comprises a heavy chain sequence of SEQ ID NO:12, 13, 132, 134, 136, 138, 140, 141, 143, 145, 146, 148, 150, 151, or 679.

[0438] In some embodiments of the invention described herein, the antibody that specifically binds to PSMA comprises a light chain sequence of SEQ ID NO:14, 15, 75, 133, 135, 137, 139, 142, 144, 147, 149, or 680.

[0439] CD33-specific antibodies

[0440] The CD33-specific antibody of the invention has one or more desired functional properties, including but not limited to high affinity binding to CD33 and / or CD3, high specificity for CD33 and / or CD3, and the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies.

[0441] In certain embodiments, the isolated monoclonal antibody or an antigen-binding fragment thereof binds to the C2 domain of CD33. In certain embodiments, the isolated monoclonal antibody or an antigen-binding fragment thereof binds to the V domain of CD33. Full-length human CD33 is provided by Uniprot P20138 (SEQ ID NO:244).

[0442] As used herein, an antibody that "specifically binds to CD33" refers to an antibody that binds to CD33 with a dissociation constant of 1×10 -7M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less or 1×10 -10 An antibody that binds to CD33 with a KD of M or less, preferably binds to human CD33, and preferably binds to the C2 domain of CD33.

[0443] The antibodies or antigen-binding fragments described herein can exist in multiple forms, but will contain one or more of the antibody CDRs shown in Tables 39 and 40.

[0444] Recombinant antibodies and antigen-binding fragments that specifically bind to CD33 are described herein. In some embodiments, the CD33-specific antibody or antigen-binding fragment is human IgG or a derivative thereof. Although the CD33-specific antibodies or antigen-binding fragments exemplified herein are human, the exemplified antibodies or antigen-binding fragments can also be chimeric.

[0445] In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided that comprise a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 39. In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided that comprise a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 39 and a light chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 40.

[0446] In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided that comprise the heavy chain variable region shown in Table 38. In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided that comprise the light chain variable region shown in Table 38.

[0447] The heavy chain variable domain and the light chain variable domain of the antibodies discussed in this section and shown in Table 38 are suitable for inclusion in bispecific constructs. For example, in some embodiments of a CD33 bispecific antibody, the effector arm is the CD3 arm. In some embodiments of a CD33×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 670, respectively. In some embodiments of a CD33×CD3 bispecific antibody, the CD3 arm comprises VH and VL that are SEQ ID NO:652 and 661, respectively. In some embodiments of a CD33×CD3 bispecific antibody, the CD3 arm comprises HC and LC that are SEQ ID NO:640 and 676, respectively.

[0448] In some embodiments of a CD33×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 773, 673, and 670, respectively. In some embodiments of a CD33×CD3 bispecific antibody, the CD3 arm comprises VH and VL that are SEQ ID NO:657 and 678, respectively. In some embodiments of a CD33×CD3 bispecific antibody, the CD3 arm comprises HC and LC that are SEQ ID NO:675 and 677, respectively.

[0449] In certain embodiments, an anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO:267, 260, 275, 270, 262, 258, 257, 281, 292, 291, 261, 269, 280, 259, 263, 264, 265, 266, 272, 277, 279, 284, or 285 or a light chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO:287, 314, 309, 301, 298, 297, 290, 332, 331, 302, 310, 320, 300, 304, 305, 306, 307, 317, 319, 324, or 325. And an anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO:257 or 258 or a light chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO:298 or 299.

[0450] IL1RAP-specific antibodies

[0451] As used herein, the terms "interleukin-1 receptor accessory protein", "IL1RAP", and "IL1-RAP" specifically include the human IL1RAP protein (SEQ ID NO:576), for example, those described in GenBank accession number AAB84059, NCBI reference sequence: NP_002173.1, and UniProtKB / Swiss-Prot accession number Q9NPH3-1 (see also Huang et al., 1997, Proc. Natl. Acad. Sci. USA. Vol. 94, No. 24, pp. 12829-12832). IL1RAP has also been referred to in the scientific literature as IL1 R3, C3orf13, FLJ37788, IL-1RAcP, and EG3556.

[0452] The antibodies or antigen-binding fragments described herein can exist in a variety of forms, but will comprise one or more of the antibody CDRs shown in Table 29.

[0453] Recombinant antibodies and antigen-binding fragments that specifically bind to IL1RAP are described herein. In some embodiments, the IL1RAP-specific antibody or antigen-binding fragment is a human IgG or a derivative thereof. Although the IL1RAP-specific antibodies or antigen-binding fragments exemplified herein are human, the exemplified antibodies or antigen-binding fragments can also be chimeric.

[0454] In some embodiments, there are provided IL1RAP-specific antibodies or antigen-binding fragments thereof that comprise a heavy chain containing CDR1, CDR2, and CDR3 of any one of the antibodies shown in Table 29. In some embodiments, there are provided IL1RAP-specific antibodies or antigen-binding fragments thereof that comprise a heavy chain containing CDR1, CDR2, and CDR3 of any one of the antibodies shown in Table 29 and a light chain containing CDR1, CDR2, and CDR3 of any one of the antibodies shown in Table 29.

[0455] In some embodiments, there are provided IL1RAP-specific antibodies or antigen-binding fragments thereof that comprise a heavy chain variable region of any one of the antibodies shown in Table 30. In some embodiments, there are provided IL1RAP-specific antibodies or antigen-binding fragments thereof that comprise a light chain variable region of any one of the antibodies shown in Table 30. In some embodiments, there are provided IL1RAP-specific antibodies or antigen-binding fragments thereof that comprise a heavy chain variable region and a light chain variable region of any one of the antibodies shown in Table 30.

[0456] The heavy chain variable domain and the light chain variable domain of the antibodies discussed in this paragraph and shown in Table 30 are suitable for inclusion in a bispecific construct, wherein the target arm is an anti-IL1RAP arm. For example, in some embodiments of an IL1RAP bispecific antibody, the effector arm is a CD3 arm. In some embodiments of an IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 670, respectively. In some embodiments of an IL1RAP×CD3 bispecific antibody, the CD3 arm comprises VH and VL that are SEQ ID NO:652 and 661, respectively. In some embodiments of an IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HC and LC that are SEQ ID NO:640 and 676, respectively.

[0457] In some embodiments of an IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 773, 673, and 670, respectively. In some embodiments of an IL1RAP×CD3 bispecific antibody, the CD3 arm comprises VH and VL that are SEQ ID NO:657 and 678, respectively. In some embodiments of an IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HC and LC that are SEQ ID NO:675 and 677, respectively.

[0458] TMEFF2-specific antibodies

[0459] The present invention provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof that binds to the membrane-proximal region of SEQ ID NO:629 of TMEFF2. The anti-TMEFF2 antibody of the present invention that binds to the membrane-proximal region of TMEFF2 is not internalized by cells. Without wishing to be bound by any particular theory, it is expected that, compared to an internalized anti-TMEFF2 antibody, the non-internalized anti-TMEFF2 antibody has an improved oncolytic effect mediated by antibody effector functions due to the lack of internalization and degradation of TMEFF2.

[0460] "Binding to the membrane-proximal region" means that 90% of the antibody epitope residues identified using hydrogen / deuterium exchange (H / D exchange) are located within the membrane-proximal region of TMEFF2. Epitope residues are those protected by the test antibody that have at least a 5% difference in deuteration level by H / D exchange. Exemplary such antibodies are TMEB675, TMEB570, TMEB674, TMEB565, TMEB762, and TMEB757 as described herein.

[0461] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof binds to the membrane-proximal region of TMEFF2 within the residues HGKCEHSINMQEPSC (SEQ ID NO:592) or DAGYTGQHCEKKDYSVL (SEQ ID NO:600). An exemplary anti-TMEFF2 antibody that binds within the residues HGKCEHSINMQEPSC (SEQ ID NO:592) is TMEB570. An exemplary anti-TMEFF2 antibody that binds within the residues DAGYTGQHCEKKDYSVL (SEQ ID NO:600) is TMEB675. TNEB675 variants TMEB762 and TMEB757 are also expected to bind to the membrane-proximal region of TMEFF2 within the residues DAGYTGQHCEKKDYSVL (SEQ ID NO:600).

[0462] In the H / D exchange assay, recombinantly expressed TMEFF2 ECD is incubated in deuterated water in the presence or absence of antibody for a predetermined time, resulting in the introduction of deuterium at exchangeable hydrogen atoms not protected by the antibody. Protease digestion of the protein is then carried out and the peptide fragments are analyzed using LC-MS. The H / D exchange assay can be performed using known protocols. An exemplary protocol is described in Example 5.

[0463] The invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment competes with a reference antibody for binding to the membrane-proximal region of TMEFF2, the reference antibody comprising the heavy chain variable region (VH) of SEQ ID NO:25 and the light chain variable region (VL) of SEQ ID NO:28, the VH of SEQ ID NO:589 and the VL of SEQ ID NO:29, the VH of SEQ ID NO:27 and the VL of SEQ ID NO:30, the VH of SEQ ID NO:589 and the VL of SEQ ID NO:31, the VH of SEQ ID NO:604 and the VL of SEQ ID NO:607, or the VH of SEQ ID NO:612 and the VL of SEQ ID NO:613.

[0464] The ability of a test antibody to compete with a reference antibody for binding to the membrane-proximal region of TMEFF2 can be assayed in vitro using well-known methods. For example, binding of an MSD Sulfo-Tag TM NHS-ester-labeled test antibody to the membrane-proximal region of TMEFF2 can be evaluated by ELISA in the presence of an unlabeled reference antibody, or competition can be demonstrated using a Bioacore assay or flow cytometry. A test antibody competes with a reference antibody for binding to TMEFF2 when it inhibits binding of the reference antibody to the membrane-proximal region of TMEFF2 by 85% or more, such as 90% or more, or 95% or more.

[0465] The invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises the following heavy chain complementarity determining regions 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining regions 1 (LCDR1), LCDR2, and LCDR3:

[0466] SEQ ID NO:582, 584, 587, 18, 588, and 22, respectively;

[0467] SEQ ID NO:583, 585, 16, 19, 21, and 23, respectively;

[0468] SEQ ID NO:582, 586, 17, 18, 588, and 24, respectively;

[0469] SEQ ID NO:583, 585, 16, 18, 588, and 22, respectively; or

[0470] SEQ ID NO:582, 584, 587, 18, 588, and 603, respectively.

[0471] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof binds to the membrane-proximal region of TMEFF2 with an equilibrium dissociation constant (K -9 ) of about 0.4×10 D M or less, where K D is measured using surface plasmon resonance in acetate buffer at pH 4.5 - 5.0 at room temperature.

[0472] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof binds to the membrane-proximal region of TMEFF2 with a K - 10 between about 0.1×10 -9 M and about 0.4×10 D M.

[0473] The affinity of an antibody for the membrane-proximal region of TMEFF2 can be determined experimentally using any suitable method. An exemplary method employs a ProteOn XPR36, Biacore 3000, or KinExA instrument, ELISA, or competitive binding assay known to those of skill in the art. If measured under different conditions (e.g., osmolarity, pH), the measured affinity of the antibody for TMEFF2 can vary. Thus, the affinity and other binding parameters (e.g., K D , K on , and K off ) are typically measured using standardized conditions and standardized buffers (such as those described herein). Those of skill in the art will understand that the internal error in affinity measurements using, for example, a Biacore 3000 or ProteOn (measured as standard deviation, SD) is typically within 5%-33% of the measured value within the typical limit of detection. Thus, when referring to K D values, the term "about" reflects the typical standard deviation in the assay. For example, the typical SD of a K -9 of 1×10 D M is at most ±0.33×10 -9 M.

[0474] The invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to the membrane-proximal region of TMEFF2 and comprises a heavy chain variable region (VH) framework derived from VH3_3-23 (SEQ ID NO:53) or VH1_1-69 (SEQ ID NO:54).

[0475] The invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to the membrane-proximal region of TMEFF2 and comprises a light chain variable region (VL) framework derived from VKI_L11 (SEQ ID NO:55) or VKIIII_A27 (SEQ ID NO:591).

[0476] The invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to the membrane-proximal region of TMEFF2 and comprises a VH framework and a VL framework derived from VH3_3-23 of SEQ ID NO:53 and VKI_L11 of SEQ ID NO:55, respectively.

[0477] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment binds to the membrane-proximal region of TMEFF2 and comprises a VH framework and a VL framework derived from VH1_1-69 of SEQ ID NO:54 and VKIII_A27 of SEQ ID NO:591, respectively.

[0478] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment binds to the membrane-proximal region of TMEFF2 and comprises a VH framework and a VL framework derived from VH1_1-69 of SEQ ID NO:54 and VKI_L11 of SEQ ID NO:55, respectively.

[0479] An antibody comprising a heavy or light chain variable region "derived from" a particular framework or germline sequence refers to an antibody obtained from a system using human germline immunoglobulin genes, such as an antibody obtained from a transgenic mouse, rat or chicken or a phage display library as discussed herein. Due to, for example, naturally occurring somatic mutations or intentional substitutions, an antibody containing a particular framework derived from a germline sequence may contain amino acid differences compared to the sequence from which it is derived.

[0480] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are SEQ ID NO:582, 584, 587, 18, 588 and 22, respectively.

[0481] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:25 and a VL of SEQ ID NO:28.

[0482] In some embodiments, VH is encoded by a polynucleotide of SEQ ID NO:39 and VL is encoded by a polynucleotide of SEQ ID NO:42.

[0483] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises an HC of SEQ ID NO:32 and an LC of SEQ ID NO:35.

[0484] In some embodiments, HC is encoded by a polynucleotide of SEQ ID NO:46 and VL is encoded by a polynucleotide of SEQ ID NO:49.

[0485] The present invention also provides a isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:583, 585, 16, 19, 21 and 23 respectively.

[0486] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises VH of SEQ ID NO:589 and VL of SEQ ID NO:29.

[0487] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:40, and VL is encoded by the polynucleotide of SEQ ID NO:43.

[0488] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises HC of SEQ ID NO:33 and LC of SEQ ID NO:36.

[0489] In some embodiments, HC is encoded by the polynucleotide of SEQ ID NO:47, and VL is encoded by the polynucleotide of SEQ ID NO:50.

[0490] The present invention also provides a isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:582, 586, 17, 18, 588 and 24 respectively.

[0491] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30.

[0492] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:41, and VL is encoded by the polynucleotide of SEQ ID NO:44.

[0493] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises HC of SEQ ID NO:34 and LC of SEQ ID NO:37.

[0494] In some embodiments, HC is encoded by the polynucleotide of SEQ ID NO:48, and LC is encoded by the polynucleotide of SEQ ID NO:51.

[0495] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:583, 585, 16, 18, 588 and 22 respectively.

[0496] In some embodiments, the isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof comprises VH of SEQ ID NO:589 and VL of SEQ ID NO:31.

[0497] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:40, and VL is encoded by the polynucleotide of SEQ ID NO:45.

[0498] In some embodiments, the isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof comprises HC of SEQ ID NO:33 and LC of SEQ ID NO:38.

[0499] In some embodiments, HC is encoded by the polynucleotide of SEQ ID NO:47, and LC is encoded by the polynucleotide of SEQ ID NO:590.

[0500] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:582, 584, 587, 18, 588 and 603 respectively.

[0501] In some embodiments, the isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof comprises VH of SEQ ID NO:604 and VL of SEQ ID NO:607.

[0502] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:618, and VL is encoded by the polynucleotide of SEQ ID NO:619.

[0503] In some embodiments, the isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof comprises HC of SEQ ID NO:614 and LC of SEQ ID NO:615.

[0504] In some embodiments, HC is encoded by the polynucleotide of SEQ ID NO:620, and LC is encoded by the polynucleotide of SEQ ID NO:621.

[0505] The present invention also provides a isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:582, 584, 587, 18, 588 and 603 respectively.

[0506] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises VH of SEQ ID NO:612 and VL of SEQ ID NO:613.

[0507] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:622, and VL is encoded by the polynucleotide of SEQ ID NO:623.

[0508] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises HC of SEQ ID NO:616 and LC of SEQ ID NO:617.

[0509] In some embodiments, HC is encoded by the polynucleotide of SEQ ID NO:624, and LC is encoded by the polynucleotide of SEQ ID NO:625.

[0510] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is a multispecific antibody.

[0511] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is a bispecific antibody.

[0512] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds to a T cell antigen.

[0513] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds to CD3.

[0514] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds to CD3ε.

[0515] The VH, VL, HCDR, LCDR, HC and LC sequences of exemplary anti-TMEFF2 antibodies of the present invention are shown in Tables 60 to 67.

[0516] Although the embodiments shown in the examples include paired variable domains, one from a heavy chain and one from a light chain, those skilled in the art will recognize that alternative embodiments may include a single heavy chain variable domain or a single light chain variable domain. A single variable domain can be used to screen variable domains capable of forming a two-domain specific antigen-binding fragment that can bind to TMEFF2. Screening can be accomplished by phage display screening methods, using, for example, the hierarchical dual combinatorial method disclosed in International Patent Publication WO1992 / 01047. In this method, a single colony containing a VH or VL chain clone is used to infect a complete library of clones encoding the other chain (VL or VH), and the resulting double-stranded specific antigen-binding domains are selected according to phage display techniques using known methods and those described herein. Thus, using the method disclosed in International Patent Publication WO1992 / 01047, the individual VH and VL polypeptide chains can be used to identify additional anti-TMEFF2 antibodies.

[0517] Bispecific anti-TMEFF2 / anti-CD3 antibodies

[0518] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the antibody binds to the membrane-proximal region of TMEFF2. Without wishing to be bound by any particular theory, a bispecific antibody that binds to the membrane-proximal region of TMEFF2 may more effectively mediate T cell-mediated tumor cell killing.

[0519] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the antibody competes with a reference antibody for binding to the membrane-proximal region of TMEFF2, the reference antibody comprising the heavy chain variable region (VH) of SEQ ID NO:25 and the light chain variable region (VL) of SEQ ID NO:28, the VH of SEQ ID NO:589 and the VL of SEQ ID NO:29, the VH of SEQ ID NO:27 and the VL of SEQ ID NO:30, the VH of SEQ ID NO:589 and the VL of SEQ ID NO:31, the VH of SEQ ID NO:604 and the VL of SEQ ID NO:607, or the VH of SEQ ID NO:612 and the VL of SEQ ID NO:613.

[0520] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is at about 0.4×10 -9An M or smaller dissociation constant (K D ) binds to the membrane-proximal region of TMEFF2, where K D is measured at room temperature in acetate buffer at pH 4.5 - 5.0 using surface plasmon resonance.

[0521] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof has a K -10 between about 0.1×10 -9 M and about 0.4×10 D M and binds to the membrane-proximal region of TMEFF2.

[0522] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the first domain comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3:

[0523] SEQ ID NO:582, 584, 587, 18, 588, and 22, respectively;

[0524] SEQ ID NO:583, 585, 16, 19, 21, and 23, respectively;

[0525] SEQ ID NO:582, 586, 17, 18, 588, and 24, respectively; o

[0526] SEQ ID NO:583, 585, 16, 18, 588, and 22, respectively; or

[0527] SEQ ID NO:582, 584, 587, 18, 588, and 603, respectively.

[0528] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 which are SEQ ID NO:662, 663, 664, 671, 673, and 690, respectively.

[0529] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the first domain comprises

[0530] VH of SEQ ID NO:25 and VL of SEQ ID NO:28;

[0531] VH of SEQ ID NO:589 and VL of SEQ ID NO:29;

[0532] VH of SEQ ID NO:27 and VL of SEQ ID NO:30;

[0533] VH of SEQ ID NO:589 and VL of SEQ ID NO:31;

[0534] VH of SEQ ID NO:604 and VL of SEQ ID NO:607; or

[0535] VH of SEQ ID NO:612 and VL of SEQ ID NO:613.

[0536] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the second domain comprises

[0537] VH of SEQ ID NO:652 and VL of SEQ ID NO:661.

[0538] In some embodiments, the second domain comprises VH of SEQ ID NO:657 and VL of SEQ ID NO:658.

[0539] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0540] the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:582, 584, 587, 18, 588, and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 690, respectively;

[0541] the first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0542] the first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:657 and VL of SEQ ID NO:678;

[0543] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:32, LC1 of SEQ ID NO:35, HC2 of SEQ ID NO:640, and LC2 of SEQ ID NO:676; and / or

[0544] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:32, LC1 of SEQ ID NO:35, HC2 of SEQ ID NO:675, and LC2 of SEQ ID NO:677.

[0545] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0546] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:583, 585, 16, 19, 21, and 23 respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 690 respectively;

[0547] The first domain comprises VH of SEQ ID NO:589 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0548] The first domain comprises VH of SEQ ID NO:589 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0549] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:640, and LC2 of SEQ ID NO:676;

[0550] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:675, and LC2 of SEQ ID NO:677;

[0551] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0552] the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:582, 586, 17, 18, 588 and 24 respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:662, 663, 664, 671, 673 and 690 respectively;

[0553] the first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0554] the first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0555] the bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:640 and LC2 of SEQ ID NO:676;

[0556] the bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:675 and LC2 of SEQ ID NO:677;

[0557] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0558] The first domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:583, 585, 16, 18, 588, and 22 respectively, and the second domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 690 respectively;

[0559] The first domain contains VH of SEQ ID NO:589 and VL of SEQ ID NO:31, and the second domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0560] The first domain contains VH of SEQ ID NO:589 and VL of SEQ ID NO:31, and the second domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0561] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment contains HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:640, and LC2 of SEQ ID NO:676;

[0562] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment contains HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:675, and LC2 of SEQ ID NO:677.

[0563] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment, which antibody or antigen-binding fragment contains a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0564] The first domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:582, 584, 587, 18, 588, and 603 respectively, and the second domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673, and 690 respectively;

[0565] The first domain comprises a VH of SEQ ID NO:604 and a VL of SEQ ID NO:607, and the second domain comprises a VH of SEQ ID NO:652 and a VL of SEQ ID NO:661;

[0566] The first domain comprises a VH of SEQ ID NO:604 and a VL of SEQ ID NO:607, and the second domain comprises a VH of SEQ ID NO:657 and a VL of SEQ ID NO:678; and / or

[0567] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:614, LC1 of SEQ ID NO:615, HC2 of SEQ ID NO:640 and LC2 of SEQ ID NO:676;

[0568] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:614, LC1 of SEQ ID NO:615, HC2 of SEQ ID NO:675 and LC2 of SEQ ID NO:677;

[0569] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0570] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are SEQ ID NO:582, 584, 587, 18, 588 and 603 respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are SEQ ID NO:662, 663, 664, 671, 673 and 690 respectively;

[0571] The first domain comprises a VH of SEQ ID NO:612 and a VL of SEQ ID NO:613, and the second domain comprises a VH of SEQ ID NO:652 and a VL of SEQ ID NO:661;

[0572] The first domain comprises a VH of SEQ ID NO:612 and a VL of SEQ ID NO:613, and the second domain comprises a VH of SEQ ID NO:657 and a VL of SEQ ID NO:678; and / or

[0573] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:616, LC1 of SEQ ID NO:617, HC2 of SEQ ID NO:640, and LC2 of SEQ ID NO:676;

[0574] The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:616, LC1 of SEQ ID NO:617, HC2 of SEQ ID NO:675, and LC2 of SEQ ID NO:677.

[0575] Embodiments :

[0576] The present invention provides the following non-limiting embodiments.

[0577] 1. An isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof, comprising:

[0578] a) a heavy chain and a light chain, the heavy chain comprising: heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO:662, HCDR2 comprising SEQ ID NO:663, and HCDR3 comprising SEQ ID NO:664, the light chain comprising: light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO:671,

[0579] LCDR2 comprising SEQ ID NO:673, and LCDR3 comprising SEQ ID NO:690;

[0580] b) a heavy chain variable region comprising SEQ ID NO:652 and a light chain variable region comprising SEQ ID NO:661;

[0581] c) a heavy chain comprising SEQ ID NO:640 and a light chain comprising SEQ ID NO:676;

[0582] d) a heavy chain and a light chain, the heavy chain comprising: HCDR1 comprising SEQ ID NO:662, HCDR2 comprising SEQ ID NO:663, and

[0583] HCDR3 comprising SEQ ID NO:664, the light chain comprising: LCDR1 comprising SEQ ID NO:773, LCDR2 comprising SEQ ID NO:673, and

[0584] LCDR3 comprising SEQ ID NO:690;

[0585] e) comprising a heavy chain variable region of SEQ ID NO: 657 and a light chain variable region of SEQ ID NO: 678; or

[0586] f) comprising a heavy chain of SEQ ID NO: 675 and a light chain of SEQ ID NO: 678. 2. An isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof, wherein said antibody or

[0587] antigen-binding fragment specifically binds cynomolgus monkey or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of about 300 nM or less.

[0588] 3. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to embodiment 2, wherein said binding affinity is about 100 nM or less.

[0589] 4. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to embodiment 2 or 3, wherein said binding affinity is measured by flow cytometry or by Proteon surface plasmon resonance using a ProteOn XPR36 system at +25 °C.

[0590] 5. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein said antibody or antigen-binding fragment has one, two, three, or four of the following characteristics:

[0591] a) binds human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 300 nM or less, and binds HEK cells expressing cynomolgus monkey CD3 with a calculated EC50 of 300 nM or less, wherein the difference in calculated EC50 between binding to CD3+ T lymphocytes and binding to HEK cells expressing cynomolgus monkey CD3 is less than 5-fold, and wherein said calculated EC50 is measured by flow cytometry in a whole cell binding assay at 0 °C;

[0592] b) binds recombinant D from humans with an equilibrium dissociation constant (K

[0593] CD3d (SEQ ID NO: 691), or binds recombinant CD3e (SEQ ID NO: 636) from humans, or binds recombinant CD3d (SEQ ID NO: 692) from cynomolgus monkeys, or binds recombinant CD3e (SEQ ID NO:

[0594] 693) from cynomolgus monkeys, wherein said K D is measured by Proteon surface plasmon resonance using a ProteOn XPR36 system at +25 °C;

[0595] c) residues 1-6 of CD3e, as determined by X-ray crystallography; or

[0596] d) activates T cells or induces CD69 expression to a level similar to cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0597] 6. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, comprising at least one substitution in the antibody constant domain, said at least one substitution comprising:

[0598] a) a heavy chain substitution of K409R, F405L or F405L and R409K;

[0599] b) a heavy chain substitution of S228P, F234A and L235A;

[0600] c) a heavy chain substitution of L234A, G237A, P238S, H268A, A330S and

[0601] P331S, wherein the antibody is of IgG1 isotype; or

[0602] d) a heavy chain substitution of S228P, wherein the antibody is of IgG4 isotype;

[0603] wherein the residues are numbered according to the EU index.

[0604] 7. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO:662, 663, 664, 671, 673 and 690 respectively.

[0605] 8. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, comprising a heavy chain variable region (VH) and a light chain variable region (VL) which are SEQ ID NO:652 and 661 respectively.

[0606] 9. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, comprising a heavy chain sequence (HC) and a light chain sequence (LC) which are SEQ ID NO:640 and 676 respectively.

[0607] 10. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising SEQ ID NO:662, 663, 664, 773, 673 and 690 respectively

[0608] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0609] 11. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising VH and VL that are SEQ ID NO: 657 and 678, respectively.

[0610] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising HC and LC that are SEQ ID NO: 675 and 677, respectively.

[0611] 13. An antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663,

[0612] 664, 671, 673, and 690, respectively.

[0613] 14. An antibody or antigen-binding fragment thereof, comprising VH and VL that are SEQ ID NO: 652 and 661, respectively

[0614] respectively.

[0615] 15. An antibody or antigen-binding fragment thereof, comprising HC and LC that are SEQ ID NO: 640 and 676, respectively

[0616] respectively.

[0617] 16. An antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663,

[0618] 664, 773, 673, and 690, respectively.

[0619] 17. An antibody or antigen-binding fragment thereof, comprising VH and VL that are SEQ ID NO: 657 and 678, respectively

[0620] respectively.

[0621] 18. An antibody or antigen-binding fragment thereof, comprising HC and LC that are SEQ ID NO: 675 and 677, respectively

[0622] respectively.

[0623] 19. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein

[0624] the antibody is human or humanized.

[0625] 20. The antibody according to embodiment 19, wherein the antibody is an IgG4 or IgG1 isotype.

[0626] 21. The antibody according to embodiment 20, comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc of the antibody.

[0627] 22. The antibody according to embodiment 18, comprising:

[0628] a) D43G, L49M, L50I, S62N, Q85E light chain substitutions;

[0629] b) D43G, V48L, L49M, L50I, S62N, Q85E, H89Y light chain substitutions;

[0630] c) R10G, R13K, V73I, R70K, T83S, L96V heavy chain substitutions;

[0631] d) Any one of the light chain substitutions D43G, V48L, L49M, L50I, S62N, Q85E or H89Y; or

[0632] e) Any one of the heavy chain substitutions R10G, R13K, V73I, R79K, T83S or L96V,

[0633] wherein the residues of the light chain substitutions are numbered according to SEQ ID No: 661, and the residues of the heavy chain substitutions are numbered according to SEQ ID No: 652.

[0634] 23. The antibody according to any one of the foregoing embodiments, wherein the antibody is bispecific or multispecific.

[0635] 24. A bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 that are SEQ ID NO: 662, 663, 664, 671, 673 and 690, respectively.

[0636] 25. The bispecific antibody according to embodiment 24, wherein the first domain and the second domain are of the IgG4 isotype, and wherein the first domain or the second domain comprises the heavy chain substitutions S228P, F234A, L235A, F405L and R409K, and the other domain of the first domain or the second domain comprises the heavy chain substitutions S228P, F234A and L235A, wherein the residues are numbered according to the EU index.

[0637] 26. The bispecific antibody according to embodiment 24, wherein the first domain and / or the second domain comprises at least one substitution in the CH3 constant domain, the CH3 constant domain comprising an F405L or an F405L and an R409K substitution, wherein the residues are numbered according to the EU index.

[0638] 27. The bispecific antibody according to embodiment 24, wherein one of the first domain or the second domain comprises an F405L heavy chain substitution and the other of the first domain or the second domain comprises a K409R heavy chain substitution, wherein the residues are numbered according to the EU index.

[0639] 28. The bispecific antibody according to embodiment 24, wherein the first domain and the second domain are of the IgG4 isotype, wherein one of the first domain or the second domain comprises an S228P heavy chain substitution and the other of the first domain or the second domain comprises an S228P, F405L and R409K heavy chain substitution, wherein the residues are numbered according to the EU index.

[0640] 29. The bispecific antibody according to claim 24, wherein the first domain comprises a VH and a VL that are SEQ ID NO: 652 and 661, respectively.

[0641] 30. The bispecific antibody according to claim 24, wherein the first domain comprises an HC and an LC that are SEQ ID NO: 640 and 676, respectively.

[0642] 31. The bispecific antibody according to claim 24, wherein the first domain comprises a VH and a VL that are SEQ ID NO: 657 and 678, respectively.

[0643] 32. The bispecific antibody according to claim 24, wherein the first domain comprises an HC and an LC that are SEQ ID NO: 675 and 677, respectively.

[0644] 33. The bispecific antibody according to claim 24, wherein the second antigen is a cell surface antigen expressed on a target cell other than an immune effector cell.

[0645] 34. The bispecific antibody according to claim 33, wherein the cell surface antigen is a tumor-associated antigen.

[0646] 35. The bispecific antibody according to any one of claims 24 to 34, wherein the second antigen is CD33, IL1RAP, PSMA or TMEFF2.

[0647] 36. The bispecific antibody according to embodiment 35, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 78, 683, 80, 81, 792, and 686, respectively.

[0648] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0649] 37. The bispecific antibody according to embodiment 33, wherein the first domain comprises VH and VL that are SEQ ID NO: 652 and 661, respectively; and wherein the second domain comprises VH and VL that are SEQ ID NO: 681 and 682, respectively.

[0650] 38. The bispecific antibody according to embodiment 33, wherein the first domain comprises HC and LC that are SEQ ID NO: 640 and 676, respectively; and wherein the second domain comprises HC and LC that are SEQ ID NO: 679 and 680, respectively.

[0651] 39. The bispecific antibody according to embodiment 33, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that are SEQ ID NO: 78, 3, 80, 81, 792, and 686, respectively.

[0652] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0653] 40. The bispecific antibody according to embodiment 33, wherein the first domain comprises VH and VL that are SEQ ID NO: 652 and 661, respectively; and wherein the second domain comprises VH and VL that are SEQ ID NO: 8 and 682, respectively..

[0654] 41. The bispecific antibody according to embodiment 33, wherein the first domain comprises HC and LC that are SEQ ID NO: 640 and 676, respectively; and wherein the second domain comprises HC and LC that are SEQ ID NO: 13 and 680, respectively.

[0655] 42. The bispecific antibody according to embodiment 33, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO: 662, 663, 664, 671, 673 and 690 respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO: 78, 79, 80, 81, 82 and 83 respectively.

[0656] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0657] 43. The bispecific antibody according to embodiment 33, wherein the first domain comprises VH and VL which are SEQ ID NO: 652 and 661 respectively; and wherein the second domain comprises VH and VL which are SEQ ID NO: 116 and 117 respectively.

[0658] 44. The bispecific antibody according to embodiment 33, wherein the first domain comprises HC and LC which are SEQ ID NO: 640 and 676 respectively; and wherein the second domain comprises HC and LC which are SEQ ID NO: 138 and 139 respectively.

[0659] 45. The bispecific antibody according to embodiment 31, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO: 662, 663, 664, 671, 673 and 670 respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 which are SEQ ID NO: 164, 165, 166, 167, 168 and

[0660] 169 respectively.

[0661] 46. The bispecific antibody according to embodiment 31, wherein the first domain comprises VH and VL which are SEQ ID NO: 652 and 661 respectively; and wherein the second domain comprises VH and VL which are SEQ ID NO: 215 and 216 respectively.

[0662] 47. The bispecific antibody according to embodiment 31, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 which are SEQ ID NO: 662, 663, 664, 671, 673, and 670 respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 which are SEQ ID NO: 349, 390, 341, 471, 513, and 555 respectively.

[0663] 48. The bispecific antibody according to embodiment 31, wherein the first domain comprises VH and VL which are SEQ ID NO: 652 and 661 respectively; and wherein the second domain comprises VH and VL which are SEQ ID NO: 267 and 306 respectively.

[0664] 49. The bispecific antibody according to embodiment 31, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 which are SEQ ID NO: 662, 663, 664, 671, 673, and 670 respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 which are SEQ ID NO: 363, 404, 445, 485, 527, and

[0665] 569 respectively.

[0666] 50. The bispecific antibody according to embodiment 31, wherein the first domain comprises VH and VL which are SEQ ID NO: 652 and 661 respectively; and wherein the second domain comprises VH and VL which are SEQ ID NO: 281 and 320 respectively.

[0667] 51. A pharmaceutical composition comprising the antibody according to any one of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0668] 52. A polynucleotide encoding the antibody according to any one of embodiments 1 to 50.

[0669] 53. A vector comprising the polynucleotide according to embodiment 52.

[0670] 54. A host cell comprising the vector according to embodiment 53.

[0671] 55. A method for preparing an antibody according to any one of embodiments 1 to 50, comprising: culturing a host cell according to embodiment 52 under conditions that allow expression of the antibody, and recovering the antibody produced by the host cell.

[0672] 56. A method for treating cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of an isolated antibody according to any one of embodiments 1 to 50 for a time sufficient to treat the cancer.

[0673] 57. The method according to embodiment 56, wherein the cancer is a solid tumor or a hematological malignancy.

[0674] 58. The method according to embodiment 57, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell renal carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disease, clear cell renal carcinoma (CCRCC), pancreatic cancer, kidney cancer, urothelial carcinoma, or hepatic metastatic adenocarcinoma.

[0675] 59. The method according to embodiment 58, wherein the prostate cancer is refractory prostate cancer, prostatic intraepithelial neoplasia, androgen-independent prostate cancer, or malignant prostate cancer.

[0676] 60. The method according to embodiment 57, wherein the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0677] 61. The method according to any one of embodiments 56 to 60, wherein the antibody is administered in combination with a second therapeutic agent.

[0678] 62. An antibody according to any one of embodiments 1 to 50, for use in therapy.

[0679] 63. An anti-idiotypic antibody that binds to an antibody according to any one of embodiments 1 to 50.

[0680] Examples

[0681] 1 De novo generation and functional characterization of anti-CD3 mAbs

[0682] 1-1 Immunization with CD3 antigen To generate CD3 monoclonal antibodies

[0683] Immunize with a proprietary vector (Aldevron, Fargo, North Dakota, USA) encoding the following: Human CD3e and human CD3d; cynomolgus CD3e and cynomolgus CD3d. Animals received alternating booster immunizations with human and cynomolgus DNA. Starting from the 6th administration, animals received an optimized vector with the same insert sequence. Cells from lymph nodes were fused with the Ag8 myeloma cell line. After IgM depletion, 40 million cells from the fusion BLW were plated on three 96-well plates. Without IgM depletion, 133 million cells from the fusion BLX were plated on nine 96-well plates.

[0684] Hybridoma supernatants from fusions BLW (magnetic bead depletion of lymphocytes) and BLX (no magnetic bead depletion of lymphocytes) were analyzed by cell-based ELISA (CELISA) of cells transiently transfected with human and cynomolgus cDNAs cloned into screening vectors as follows: pOPT-CD3e-hum-epsilon-TCE.OMT + pOPT-CD3d-hum-delta.OMT, 1:1 (pOPT-CD3e / d-hum-mix) and pcDNA3.1-CD3e-cyn-delta + pcDNA3.1-CD3d-cyn-delta, 1:1 (pcDNA3.1-CD3e / d-cyn-mix). Human and cynomolgus cDNA sequences (and corresponding amino acid sequences) are provided in Table 4. For the CELISA negative control, untransfected mammalian cells were incubated with the hybridoma supernatant and detected with a Bethyl secondary antibody. For the CELISA transfection control, mammalian cells transfected with the above constructs were detected with an anti-tag antibody.

[0685] Hybridoma supernatants were further analyzed by flow cytometry (FACS) of CD3-positive and CD3-negative Jurkat cells: Jurkat CD3+ (E6-1) and Jurkat CD3- (J.RT3-T3.5). For the FACS negative control, CD3-negative Jurkat cells (J.RT3-T3.5) were incubated with dilution buffer and detected with Southern anti-rat Ig HRP and Bethyl anti-rat IgG1, 2a, 2b, 2c-HRP secondary antibodies.

[0686] Antibodies in the hybridoma supernatant from hybridomas of fusions BLW and BLX, specificities for the human and cynomolgus monkey CD3e / d complex presented on transiently transfected cells (or, for the human CD3e / d complex presented on Jurkat CD3+(E6-1) cells), were confirmed by performing CELISA on transiently transfected cells and testing in FACS on the Jurkat cell line (Table 3). No significant signal was detected in any of the experimental samples used as negative controls.

[0687] Table 3: Specificity of individual hybridoma supernatants tested by cell-based ELISA (upper panel) and by flow cytometry (lower panel). CELISA values represent relative fluorescence units (rfu) for each sample. FACS values represent the geometric mean (geometric mean) of relative fluorescence intensity for each sample Table 4. CD3 sequences used for immunization Human CD3d 。

[0688]

[0689]

[0690] NP_000723.1 (www.uniprot.org / uniprot / P04234) (SEQ ID NO:691)

[0691] Human CD3e (NP_000724.1 (www.uniprot.org / uniprot / P07766) (SEQ ID NO:636) Cynomolgus monkey CD3d XP_001097302 (www.uniprot.org / uniprot / Q95LI8) (SEQ ID NO:692) Cynomolgus monkey CD3e CD3e+TCE (www.uniprot.org / uniprot / Q95LI5) (SEQ ID NO:693) 1-2 Cloning of anti-CD3 antibodies Table 5A. Cloned peptide IDs and protein IDs

[0692] Clone ID

[0693] Anti-human CD3 antibodies were generated in OmniRats (OMT, Palo Alto, California, USA). The variable region (“V-region”) sequences of these clones were extracted from the genomic sequences and analyzed. All the sequences obtained were of human IgG heavy chain or λ light chain, and these sequences, especially LC, showed high homology. Alignment of the sequences with the germline showed some mutations in the framework (Figure 1). The V-region DNA sequences were synthesized and cloned into mammalian expression vectors, the heavy chain sequences were synthesized and cloned into human IgG1 vectors, and the light chain sequences were synthesized and cloned into human λ vectors. The sequences are shown in Tables 6 and 7. Protein identifiers were assigned to seven mAbs (Table 5A).

[0694] CD3B312 was selected as the most representative clone, and the heavy chain sequence was cloned into human IgG1σ and IgG4 PAA with S228P, F234A, L235A mutations and the assigned protein identifier, as shown in Table 5B. These were used to generate bispecific antibodies and the T cell redirection function was demonstrated by cytotoxicity.

[0695] HC peptide ID

[0696] LC peptide ID Protein ID BLW-2B4 CD3H218 CD3L123 CD3B311 BLW-2E6 CD3H219 CD3L124 CD3B312 BLW-3B4 CD3H218 CD3L125 CD3B313 BLX-1F8 CD3H220 CD3L126 CD3B314 BLX-2E9 CD3H221 CD3L124 CD3B315 BLX-3F4 CD3H222 CD3L124 CD3B316 BLX-3G8 CD3H223 CD3L124 CD3B317 Table 5B. CD3B312 was selected as the most representative clone, and the heavy chain sequence was cloned into human IgG1σ and IgG4PAA with mutations and specified protein identifiers IgG1

[0697] IgG1σ IgG4PAA 。

[0698] CD3B312 CD3B337 CD3B373 Table 6. Heavy chain and light chain sequences of 7 monoclonal CD3 antibodies Table 7A. VH and VL sequences of HC and LC isotypes of 7 out of 9 monoclonal CD3 antibodies from the first batch above (see Table 3). All HC isotypes are huIgG1_G1m(17). ​

[0699] ​

[0700]

[0701]

[0702]

[0703]

[0704]

[0705] ​ ​

[0706] All LC isotypes are huLambda 2 。

[0707]

[0708]

[0709] Table 7B. CDR sequences of the HC and LC isotypes of 7 out of 9 monoclonal CD3 antibodies from the first batch above (see Table 3). Sequences are defined according to Kabat. All HC isotypes are huIgG1_G1m(17). All LC isotypes are huLambda2.

[0710]

[0711]

[0712] 1-3 Screening of hybridomas that bind to purified human and cynomolgus macaque T cells

[0713] Design cell-based binding assays for the evaluation of individual rat hybridoma supernatants with human ( Figure 2 ) and cynomolgus monkey ( Figure 3) Binding ability of purified CD3+ T lymphocytes. T cells were counted, diluted to 1×10^6 cells / mL, and incubated with 0.5 μl / mL of Live / Dead Fixable Green Dead Cell Stain (Life Technologies, L-2301). Next, the cells were aliquoted into a U-bottom plate (Falcon 353077) at 100 μl / well (1×10^5 cells / well). The plate was centrifuged at 300 g for 5 minutes to pellet the cells and the supernatant was removed. The plate was vortexed briefly to resuspend the cells. The hybridoma supernatant was diluted to 4.5 μg / mL in FACS staining buffer (BSA, BD Biosciences 554657), and then serially diluted 6-fold at a 1:3 dilution to a minimum concentration of 0.006 μg / mL. A mouse anti-human CD3 positive control (SP34-2, BD Biosciences 551916) and a negative isotype control (mouse IgG1 BD Biosciences 556648) were also diluted to 4.5 μg / mL. 50 μl of each sample was added to the T cells and incubated at 4 °C for 1 hour. The cells were washed once with staining buffer and 50 μl of secondary AF647 goat anti-mouse IgG (Life Technologies, A21235) or AF647 goat anti-rat IgG (Life Technologies, A21247) was added at 10 μg / mL according to the appropriate species (anti-rat for hybridoma samples, anti-mouse for control antibodies). The plate was incubated at 4 °C for 45 minutes and washed twice with staining buffer. The cells were resuspended in 25 μl of running buffer (staining buffer + 1 mM EDTA (Life technologies, AM9260G) + 0.1% pluronic F-68 (Life Technologies 24040-032)) and read on an Intellicyt system (Intellicyt Corp.). The results are shown in Figure 2 and Figure 3 as shown in

[0714] In other experiments, purified human T cells were seeded at 1.1×10^5 cells / well in U-bottom plates. The plates were centrifuged at 300 g for 5 minutes to pellet the cells and the supernatant was removed. The plates were briefly vortexed to resuspend the cells. Hybridoma supernatants were diluted to 30 μg / mL in FACS staining buffer (BSA, BD Biosciences 554657), then serially diluted 11-fold at a 1:3 dilution to a lowest concentration of 0.00017 μg / mL. 50 μl of each sample was added to the T cells and incubated for 1 hour at 4 °C. The cells were washed once with staining buffer and 50 μl of secondary Dylight 650 goat anti-rat IgG (Bethyl, A110-239D5) was added at 10 μg / mL. The plates were incubated for 1 hour at 4 °C and washed twice with staining buffer. The cells were resuspended in 30 μl of FACS buffer and read on a Hypercyte flow cytometer (Intellicyt Corporation). Representative dose-response curves of anti-CD3 clones binding to primary human T cells are shown in Figure 5 below.

[0715] Representative competitive binding curves with SP34-2 (a commercially available anti-human CD3 antibody with a known epitope and cross-reactive with cynomolgus monkey CD3) are shown in Figure 6 below. Initial screening results are summarized in Table 8. Six clones showed positive binding and also competed with SP34-2 for binding to primary human T cells.

[0716] 1-4 Competition assay with the commercially available CD3 antibody SP34-2

[0717] The ability of the hybridoma supernatants to compete with the commercially available anti-human CD3 antibody SP34-2, which has a known epitope and cross-reacts with cynomolgus monkey CD3, was also evaluated. First, a concentration titration curve of AF488-fluorescently labeled SP34-2 (BD, 557705) was performed to determine a fixed concentration of SP34-2 for use in the following competition assay. Briefly, human purified T cells were diluted to 1×10^6 cells / mL in PBS. Fc blocker (TruStain human Fc blocker, Biolegend, 422302) was added at 5 μL / 100 μL cells and the cells were seeded at 100 μL / well into a U-bottom plate. AF488 SP34-2 and AF488-labeled isotype control (AF488 mouse IgG1, BD, 400129) were serially diluted from 50 μg / mL to 0.049 μg / mL in a 1:2 dilution scheme. The plate was centrifuged at 300 x g for 5 minutes to pellet the cells and the supernatant was removed. The plate was gently vortexed briefly to resuspend the cells. 50 μL of each AF488 SP34-2 dilution was added to the cells and incubated for 1 hour at 4 °C. The plate was washed twice with staining buffer and once with running buffer (staining buffer + 1 mM EDTA (Life technologies, AM9260G) + 0.1% pluronic F-68 (Life Technologies 24040-032)). The cells were resuspended in 25 μL of running buffer and read on an HTFC screening system (IntelliCyt Corporation). Based on the dose response curve, a fixed concentration of 2 μg / mL SP34-2 was selected for the competition assay.

[0718] The assay demonstrated that seven hybridomas that bind to purified T cells compete with SP34-2 for binding to human T cells ( Figure 4 ). Control antibodies were included in the competition assay. Unlabeled mouse anti-human CD3, SP34-2 antibody, and mouse anti-human CD3, UCHT1 antibody were used as positive controls, and rat IgG and mouse isotype were used as negative controls for the AF488-labeled SP34-2. Purified human T cells were diluted to a concentration of 1×10^6 cells / mL in PBS. Fc blocker (TruStain human Fc blocker, Biolegend, 422302) was added at 5 μL / 100 μL cells and Live / Dead Fixable Far Red dead cell stain (LifeTechnologies L10120) was added at 0.5 μL / mL cells and the cells were incubated for 15 minutes at 4 °C. Next, 10 5Cells / well (1×10^5 cells / well) were aliquoted into 96-well U-bottom plates (Falcon 353077). The plates were centrifuged at 300 x g for 5 minutes to pellet the cells and the supernatant was removed. The plates were gently vortexed briefly to resuspend the cells. Hybridoma supernatants and control antibodies were diluted to 2-fold the desired final concentration in FACS staining buffer (BSA, BD Biosciences 554657). 35 μL of 2X hybridoma supernatant and control antibody were mixed with 35 μL of 2X AF488 SP34-2 (4 μg / mL) to obtain the desired 1X concentration of hybridoma supernatant, 1X concentration of control antibody, and 2 μg / mL of AF488 SP34-2. Hybridoma supernatants and control antibodies were assayed using a 7-point titration over a range of concentrations. Hybridoma supernatants were assayed from 200 μg / mL to 0.08 μg / mL, and control antibodies were assayed from 100 μg / mL to 0.04 μg / mL. 50 μL of 1X hybridoma supernatant or 1X control antibody with 2 μg / mL AF488 SP34-2 was added to the T cells and incubated at 4 °C for 2 hours. The plates were washed twice with staining buffer and once with running buffer (staining buffer + 1 mM EDTA (Life technologies, AM9260G) + 0.1% pluronic F-68 (Life Technologies 24040-032)). The cells were resuspended in 25 μL running buffer and read on an HTFC screening system (IntelliCyt Corporation). Representative competitive binding curves with SP34-2 (a commercially available anti-human CD3 antibody with a known epitope and cross-reactive with cynomolgus monkey CD3) are shown in Figure 4 and Figure 6 . Initial screening results are summarized in Table 8. Six clones showed positive binding and also competed with SP34-2 for binding to primary human T cells.

[0719] As Figure 4 shown, seven antibodies competed with SP34-2 in a similar curve. The right shift of the curve relative to control SP34-2 indicates weaker binding affinity. As expected, the isotype control rat IgG did not compete with SP34-2.

[0720] Table 8. Summary of anti-CD3 antibodies that bind to primary human T cells. Anti-CD3 clones BLX-4E5, BLX-5H7, BLX-8B4, BLX-8B6, BLX-8G8, and BLW-1E3 are positive for binding to human T cells and compete with SP34-2 for binding

[0721]

[0722]

[0723] 1-5 Screening of hybridoma hits for T cell activation, as measured by CD69 upregulationUse assays based on primary human and cynomolgus monkey T cells to determine the ability of hybridoma hits to activate T cells. This is achieved by coating antibodies onto plates to mimic the crosslinking effect of TCR activation. Upon activation, T cells are known to upregulate the surface expression of the protein CD69. This experiment is conducted by coating 50 μl of a 10 μg / ml antibody preparation with an unknown sample or a control (positive control: internal, Okt-3 BISB264.002, BD Bioscience SP-34-2#551916; negative control: anti-CD20, internal BISB266.004) onto a 96-well plate (Costar#3361). The plate is incubated overnight at 4 °C. The next day, the plate is washed twice with PBS. Frozen primary T cells (human primary T cells from Biological Specialities or Hemacare; cynomolgus monkey primary T cells from WorldWidePrimate) are thawed, the viability is counted and resuspended at 2×10 6 cells / ml in RPMI 1640 medium (Gibco#11875, with 10% HI FBS (Gibco#10062)). 100 μl of the cells are added to the plate and incubated overnight (about 16 hours) at 37 °C, 5% CO 2 2. The next day, the plate is centrifuged at 1300 rpm for 3 minutes to pellet the cells and the supernatant is discarded. The cells are washed once in PBS and centrifuged as before. A 2.5% solution of Live / Dead green fixable stain (Life Technologies#L23101) in PBS, 10 μl, is added to each well and incubated for 10 minutes at room temperature and in the dark. Next, a 1% solution of anti-CD69 AF488 (Biolegend#310916 lot number B125271) in FACS buffer (BD Biosciences#554657), 50 μl, is added and the plate is incubated at 4 °C for 45 minutes. The plate is washed twice by pelleting the cells as before, discarding the supernatant and resuspending in 150 μl of FACS buffer. After the last wash, the cells are resuspended in 150 μl of FACS buffer and read on a FACS Canto. As Figure 19As shown, the positive controls cOkt3 and SP34-2 induced upregulation of CD69 on human T cells, as indicated by the mean fluorescence intensity of anti-CD69 staining measured. Only SP34-2 induced CD69 expression in cynomolgus monkey T cells because it binds to a CD3 sequence region conserved from monkey to human. The OKT3 anti-CD3 clone does not bind cynomolgus monkey CD3 and does not induce CD69 upregulation. The negative control in both human and cynomolgus monkey T cells was anti-CD20, which is not expressed on T cells. Among the hybridoma clones tested for T cell activation, several induced CD69 expression to a similar extent as the positive controls, namely 2B4, 2E6, 3B4, 1F8, 2E9, 3F4, 3G8, 4E5, 5H7, 8B4, 8G8, and 1F1. Most of them, except 5H7, 8B4, and 8G8, also bound and activated cynomolgus monkey T cells.

[0724] 1-6 Framework engineering of BLW-2E6

[0725] Compared to the human immunoglobulin germline sequences, the clones showed high homology and carried framework mutations ( Figure 1A and Figure 1B ). Clone 2E6 was chosen to adapt to the standard framework sequence. All 6 mutations on the HC and 7 mutations on the LC were mutated back to the human germline sequence either individually or in combination (Table 9). The synthetic mutant V region DNA sequences were cloned into the same mammalian expression vector as their parental constructs. The HC and LC constructs were paired in a matrix format to produce proteins carrying single or combined mutations, and the protein activities were tested. V48 on the LC could not be mutated back to the germline. All other reverse mutations were not critical but reduced the activity to some extent.

[0726] Table 9. BLW-2E6 framework variants .

[0727]

[0728]

[0729] The framework mutations were engineered into a hybridoma clone BLW-2E6, resulting in 80 mutant clones, some of which are shown in Table 10. The binding of the 80 mutant clones to primary human T cells was determined ( Figure 7 and Figure 8) T cells were counted, diluted to 1×10^6 cells / mL, and incubated with 5 μL of Fc blocker (TruStain human Fc blocker, Biolegend, 422302) / 100 μL of cells and 0.5 μl / mL of Live / Dead Fixable Green Dead Cell Stain (Life Technologies, L-2301) / 100 μL of cells. Next, the cells were aliquoted into 96-well U-bottom plates (Falcon 353077) at 100 μL / well (1×10^5 cells / well). The plates were centrifuged at 300 x g for 5 minutes to pellet the cells and the supernatant was removed. The plates were gently vortexed briefly to resuspend the cells. The hybridoma supernatants were diluted to 7.5 μg / mL, 1.5 μg / mL, 0.3 μg / mL, and 0.06 μg / mL in FACS staining buffer (BSA, BD Biosciences 554657). 50 μL of each sample was added to the T cells and incubated at 4 °C for 1 hour. The cells were washed once with staining buffer and 50 μL of 5 μg / mL secondary AF647 goat anti-human IgG F(ab')2 (Jackson ImmunoResearch catalog 109-605-097) was added to the cells. The plates were incubated at 4 °C for 45 minutes, washed twice with staining buffer and once with running buffer (staining buffer + 1 mM EDTA (Life technologies, AM9260G) + 0.1% pluronic F-68 (Life Technologies 24040-032)). The cells were resuspended in 25 μL of running buffer and read on an HTFC screening system (IntelliCyt Corporation). The results showed that the change in LC at position 48 eliminated binding, such that the mutation did not proceed. A slight decrease in binding was observed in HC, where all positions reverted to the germline (CD3H231).

[0730] 1-7 C91 scanning of the BLW-2E6 LC

[0731] Cloning of 2E6 and its derivatives showed poor expression and protein aggregation was observed. A residue C91 in the light chain was predicted to have a risk of post-translational modification (PTM), and was mutated to all other possible amino acids to improve protein stability (Table 10). The synthetic mutant V-region DNA sequence was cloned into the same human λ expression vector as its parental construct. SPR results showed that the change to valine or leucine at position 91 did not significantly alter the binding affinity. This change was also incorporated into the wild-type sequence and the above framework adaptation, resulting in antibodies CD3B376 (CD3H219 / CD3L150) and CD3B450 (CD3H231 / CD3L197). CD3B376 and CD3B450 were cloned as IgG4PAA (IgG4 with S228P, F234A, L235A mutations).

[0732] The sequence information of CD3B376 is provided as follows :

[0733] CD3H219 HC amino acid sequence (SEQ ID NO: 640) :

[0734] QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0735] CD3H219 HC nucleic acid sequence (SEQ ID NO: 712)

[0736] caggtgcagctgcagcagtctggccctagactcgtgcggccttcccagaccctgtctctgacctgtgccatctccggcgactccgtgttcaacaacaacgccgcctggtcctggatccggcagagcccttctagaggcctggaatggctgggccggacctactaccggtccaagtggctgtacgactacgccgtgtccgtgaagtcccggatcaccgtgaaccctgacacctcccggaaccagttcaccctgcagctgaactccgtgacccctgaggacaccgccctgtactactgcgccagaggctactcctcctccttcgactattggggccagggcaccctcgtgaccgtgtcctct

[0737] CD3L150 LC amino acid sequence (SEQ ID NO: 676) :

[0738] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0739] CD3L150 LC nucleic acid sequence (SEQ ID NO: 713) :

[0740] cagtctgctctgacccagcctgcctccgtgtctggctctcccggccagtccatcaccatcagctgtaccggcacctcctccaacatcggcacctacaagttcgtgtcctggtatcagcagcaccccgacaaggcccccaaagtgctgctgtacgaggtgtccaagcggccctctggcgtgtcctccagattctccggctccaagtctggcaacaccgcctccctgaccatcagcggactgcaggctgaggaccaggccgactaccactgtgtgtcctacgctggctctggcaccctgctgtttggcggaggcaccaagctgaccgtgctg

[0741] VH amino acid sequence of CD3H219 (SEQ ID NO: 652) :

[0742] qvqlqqsgprlvrpsqtlsltcaisgdsvfnnnaawswirqspsrglewlgrtyyrskwlydyavsvksritvnpdtsrnqftlqlnsvtpedtalyycargysssfdywgqgtlvtvss

[0743] VL amino acid sequence of CD3L150 (SEQ ID NO: 661) :

[0744] qsaltqpasvsgspgqsitisctgtssnigtykfvswyqqhpdkapkvllyevskrpsgvssrfsgsksgntasltisglqaedqadyhcVsyagsgtllfgggtkltvl

[0745] HCDR1 of CD3H219 (SEQ ID NO: 662) :NNNAAWS

[0746] HCDR2 of CD3H219 (SEQ ID NO: 663) :

[0747] RTYYRSKWLYDYAVSVKS

[0748] HCDR3 of CD3H219 (SEQ ID NO: 664) :GYSSSFDY

[0749] LCDR1 of CD3L150 (SEQ ID NO: 671) :TGTSSNIGTYKFVS

[0750] LCDR2 of CD3L150 (SEQ ID NO: 673) :EVSKRPS

[0751] LCDR3 of CD3L150 (SEQ ID NO: 690) : VSYAGSGTLL

[0752] The sequence information of CD3B450 is provided as follows :

[0753] CD3H231 HC amino acid sequence (SEQ ID NO: 675) :

[0754] QVQLQQSGPGLVKPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0755] CD3H231 HC nucleic acid sequence (SEQ ID NO: 714) :

[0756] caggtgcagctgcagcagagcggccccggcctggtcaagcccagccagaccctgagcctgacctgcgccatcagcggcgacagcgtgttcaacaacaacgccgcctggtcctggatccgccagagccccagccgcggcctggagtggctgggccgcacctactaccgcagcaagtggctgtacgactacgccgtgtccgtgaagtcccgcatcaccatcaaccccgacaccagcaagaaccagttctccctgcagctgaacagcgtgacccccgaggacaccgccgtgtactactgcgcccgcggctacagcagcagcttcgactactggggccagggcaccctggtcaccgtgtccagc

[0757] CD3L197 LC amino acid sequence (SEQ ID NO: 677) :

[0758] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0759] CD3L197 LC nucleic acid sequence (SEQ ID NO: 715) :

[0760] Cagtctgctctgacccagcctgcctccgtgtctggctctcccggccagtccatcaccatcagctgtaccggcacctcctccaacatcggcacctacaagttcgtgtcctggtatcagcagcaccccggcaaggcccccaaagtgatgatctacgaggtgtccaagcggccctccggcgtgtccaacagattctccggctccaagtccggcaacaccgcctccctgacaatcagcggactgcaggccgaggacgaggccgactactactgtgtgtcctacgccggctctggcaccctgctgtttggcggcggaacaaagctgaccgtgctg

[0761] VH amino acid sequence of CD3H231 (SEQ ID NO: 657) :

[0762] qvqlqqsgpglvkpsqtlsltcaisgdsvfnnnaawswirqspsrglewlgrtyyrskwlydyavsvksritinpdtsknqfslqlnsvtpedtavyycargysssfdywgqgtlvtvss

[0763] VL amino acid sequence of CD3L197 (SEQ ID NO: 678) :

[0764] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCVSYAGSGTLLFGGGTKLTVL

[0765] HCDR1 of CD3H231 (SEQ ID NO: 662) : NNNAAWS

[0766] HCDR2 of CD3H231 (SEQ ID NO: 663) :

[0767] RTYYRSKWLYDYAVSVKS

[0768] HCDR3 of CD3H231 (SEQ ID NO:664) : GYSSSFDY

[0769] LCDR1 of CD3L197 (SEQ ID NO:671) : TGTSSNIGTYKFVS

[0770] LCDR2 of CD3L197 (SEQ ID NO:673) : EVSKRPS

[0771] LCDR3 of CD3L197 (SEQ ID NO:690) : VSYAGSGTLL

[0772] Table 10. Engineered variants of BLW-2E6 LC measured by C91 scan

[0773] C91 scan Peptide ID CD32E6LC CD3L124 CD32E6LC, C91S CD3L146 CD32E6LC, C91G CD3L147 CD32E6LC, C91E CD3L148 CD32E6LC, C91D CD3L149 CD32E6LC, C91V CD3L150 CD32E6LC, C91A CD3L151 CD32E6LC, C91R CD3L152 CD32E6LC, C91K CD3L153 CD32E6LC, C91N CD3L154 CD32E6LC, C91M CD3L155 CD32E6LC, C91I CD3L156 CD32E6LC, C91T CD3L157 CD32E6LC, C91W CD3L158 CD32E6LC, C91Y CD3L159 CD32E6LC, C91L CD3L160 CD32E6LC, C91F CD3L161 CD32E6LC, C91Q CD3L162 CD32E6LC, C91H CD3L163 CD32E6LC, C91P CD3L164

[0774] Binding of BLW-2E6 CD3 mAb to hCD3ε construct measured by ProteOn SPR 1-8

[0775] Binding of BLW-2E6 anti-CD3 mAb with point mutations on the light and / or heavy chains to recombinant human CD3ε(1-27) peptide with a C-terminal Tencon25 fusion (produced by Janssen, designated hCD3ε(1-27)-Tn25) was measured by ProteOn SPR (Bio-Rad). Goat anti-human Fc IgG (Jackson Immunoresearch, catalog number 109-005-098) was directly immobilized at 30 μg / mL via amine coupling in acetate buffer (pH 5.0) on all 6 ligand channels vertically oriented on a GLC sensor chip (Bio-Rad, catalog number 176-5011) at a flow rate of 30 μL / min in PBS containing 0.005% Tween-20. The immobilization density averaged approximately 6000 response units (RU) with less than 5% variation between different channels. Five different mAbs in a vertical ligand orientation were captured on the anti-human Fc IgG surface at 1.5 μg / ml (1000 RU to 1250 RU), with the 6th ligand channel serving as a ligand-free surface control. In a 3-fold dilution series at 5-fold concentrations, 1 μM concentration of hCD3ε(1-27)-Tn25 was flown in as the analyte to bind to the captured mAbs in a horizontal orientation. A 6th buffer sample was also injected to monitor the dissociation of the captured mAbs and baseline stability. The dissociation phase of all concentrations of hCD3ε(1-27)-Tn25 was monitored for 30 minutes at a flow rate of 100 μL / min. The binding surface was regenerated for the next interaction cycle using an 18-second pulse of 0.8% phosphoric acid to remove the antigen and bound mAbs. The raw data was processed by subtracting two sets of reference data from the response data: 1) inter-point signal to correct for non-specific interactions between the Ag and the empty chip surface; 2) buffer channel signal to correct for baseline drift due to dissociation of the captured mAb surface over time. The processed data for each mAb at all concentrations was globally fit to a 1:1 simple Langmuir binding model to extract estimates of the kinetic (k on , k off ) and affinity (K D ) constants. The results are provided in Figure 11 .

[0776] Table 11. Summary of kinetics / affinity of BLW-2E6 variants binding to hCD3ε(1-27)-Tn25 (N = 1)

[0777]

[0778] N / A = Not Specified

[0779] Binding of anti-CD3 monoclonal antibodies to human T cells 1-9

[0780] After hybridization with the antigen-specific targeting arm, the in vitro binding affinity of CD3B376 and CD3B450 to human T cells was determined by flow cytometry. A preliminary study of human T cells was performed to determine the saturation binding constant (KdT) of the anti-CD3 tracer molecule. Then, a fixed concentration of the tracer ([T]) was used with titrated concentrations of the test mAb in a competitive binding assay. The IC50 (concentration at which 50% inhibition is achieved) value of the test molecule was used to determine the binding affinity (K d ) using the following equation: K d = IC50 / (1 + ([T] / K d T)). The saturation binding constant (K d T) of the tracer: commercially available AlexaFluor488 SP34-2 anti-CD3 (BioScience #557705) (data not shown) was determined using five human donors.

[0781] Determine the saturation binding constant (K d T)

[0782] Method: Human Pan T cells were cryopreserved in a nitrogen tank until use. The T cells were thawed, washed with PBS, resuspended in FACS staining buffer, counted (noting viability), and resuspended at 0.5 × 10^6 cells / mL. The Far Red Live / Dead stain (Life Technologies, AKA Invitrogen #L34974) (50 μL of DMSO into the vial) was added at 1 μL / 1 × 10^6 cells; and the FcR blocker (Miltenyi Biotec #130-059-901) (1 mL of 1:20 dilution / 0.5 × 10^6 cells) was added to the cells, each for 10 minutes. The cells were seeded at 50,000 cells / well and washed. Increasing concentrations of AlexaFluor488 SP-34 anti-CD3 were added to the T cells and kept at 4°C for 2 hours. The cells were washed to remove unbound antibody, fixed for 15 minutes, washed, and resuspended in FACS staining buffer containing 1 mM EDTA.

[0783] Binding was measured using an iQue Intellicyte flow cytometer. Gating was performed on the T cell population, then on single cells, and then on live cells (FL4). The geometric mean fluorescence (FL1) of each well was determined.

[0784] The mean fluorescence intensity values collected were plotted as a function of antibody molecule concentration and analyzed in a one-site binding analysis (total binding) using Prism software ( Figure 9 ). The software calculated the corresponding K dvalue that describes the binding of an antibody molecule to a receptor (CD3 on human Pan T cells), and this binding follows the law of mass action. The formula is as follows: Y = (B max × X) / (K d + X); where: Bmax is the maximum binding; K d is the ligand concentration required to achieve half-maximal binding.

[0785] Result: The K d value for each donor was derived and the average value was obtained. The saturation binding constant (K d T) for human T cells was derived as 5.6 ± 1.0 nM (n = 4), and it was used in the aforementioned formula to determine the K d binding affinity.

[0786] Determination of binding affinity of anti-CD3 mAb by competition assay

[0787] Method: Competitive binding studies were performed using a bivalent antibody against CD3:

[0788] · Bivalent anti-CD3: CD3B376 and CD3B450( Figure 10 )

[0789] Human Pan T cells were used to determine the binding affinity of the test mAb. The tracer used was the commercially available AlexaFluor488 SP-34 anti-CD3 (BioScience #557705) and the saturation binding constant of this tracer was as described above.

[0790] The T cells were cryopreserved in nitrogen tanks until use. The T cells were thawed, washed with PBS, resuspended in FACS staining buffer, counted, the viability was noted, and resuspended at 0.5 × 10 6 cells / mL. The Far Red Live / Dead stain (Life Technologies, AKA Invitrogen #L34974) (50 μL of DMSO into the vial) was added at 1 μL / 1 × 10^6 cells; and the FcR blocker (Miltenyi Biotec #130-059-901) (1 mL of 1:20 dilution / 0.5 × 10^6 cells) was added to the cells, each for 10 minutes. The cells were seeded at 50,000 cells / well and washed.

[0791] The mAb (and isotype control) was serially diluted 1:2 from an initial concentration of 1000 μg / mL or 200 μg / mL (2X), and mixed with a fixed concentration of tracer (5 μg / mL; 2X) to obtain a 1X concentration. Thus, the final (1X) concentration of the tracer was 2.5 μg / mL = 16.6 nM. This mixture was added to T cells and kept at 4 °C for 2 hours. The cells were then washed to remove unbound antibody, fixed for 15 minutes, washed, and resuspended in FACS staining buffer containing 1 mM EDTA.

[0792] Binding was measured using an iQue Intellicyte flow cytometer. Gating was performed on the T cell population, then on single cells, and then on live cells (FL4). The geometric mean fluorescence (FL1) of each well was determined. The mean fluorescence intensity values collected were plotted as a function of log antibody molecule concentration (converted to nM) and analyzed in a sigmoidal dose response (variable slope) using Prism software to derive the EC50 / IC50 values (in nM). The binding affinity (K d ) was derived using the following formula: K d = IC50 / (1 + ([T] / K d T)). Where: K d is the affinity of the competitor (unlabeled molecule); the IC50 of the test compound (in nM); [T] is the concentration of the tracer (16.6 nM); K d T is the K d of the tracer determined by saturation binding (5.6 nM for human).

[0793] Generation of monoclonal antibodies and bispecific antibodies

[0794] The bispecific CD3 antibodies of the invention can be generated by controlled Fab arm exchange (FAE) as described in the following: Labrijn et al., 2013, PNAS, Vol. 110, No. 13: pp. 5145-5150; PCT Publication WO 2011 / 131746; or Labrijn et al., 2014, Nat Protoc, Vol. 9, No. 10: pp. 2450-2463. Briefly, in this in vitro method, two full-length parental bivalent antibodies are provided, each containing a mutation in the antibody CH3 region that favors heterodimer formation, resulting in a bispecific antibody containing half arms from each parental antibody. Mutations that can be used to promote heterodimer formation are F405L in one parental antibody and R409K in the other parental antibody for IgG1 antibodies, or F405L and K409R in one parental antibody while retaining the wild-type CH3 for IgG4 antibodies.

[0795] Under the action of the CMV promoter, the monospecific antibody is expressed in the HEK cell line.

[0796] The parental antibody was purified using a Protein A column with an elution buffer of 100 mM NaAc pH 3.5 and a neutralization buffer of 2 M Tris pH 7.5 and 150 mM NaCl. The mab was desalted using a PD10 (Sephadex G25M) column and then dialyzed into D-PBS buffer at pH 7.2.

[0797] After purification, the parental antibody was mixed in 75 mM cysteamine-HCl under reducing conditions and incubated at 31 °C for 4 h. The recombination reaction was based on a molar ratio, where a set amount of the target parental (e.g., 10 mg or approximately 71.8 nanomoles) was combined with the CD3 antibody (e.g., approximately 67.8 nanomoles), with the target parental antibody added in an amount 6% higher than the CD3 antibody. The recombinant was then dialyzed against PBS to remove the reducing agent. The bispecific antibody reaction was carried out with an excess of the target parental antibody (ratio) to minimize the amount of unreacted CD3 parental antibody remaining after recombination. After partial reduction of the parental mAb, the reducing agent was removed by overnight dialysis into PBS.

[0798] Results: Figure 10 Inhibitory curves showing the competition of the bivalent and monovalent anti-CD3 constructs CD3B376 and CD3B450 for binding to the AlexaFluor488 SP-34 anti-CD3 tracer antibody are shown. Increasing concentrations of the test anti-CD3 antibody reduced the binding of the AlexaFluor488 tracer antibody, thereby reducing the mean fluorescence intensity (MFI). IC50 values were generated and the K d affinity was calculated using the aforementioned formula and summarized in Table 13.

[0799] The CD3B376 binding site is more tightly bound than both the bivalent and monovalent forms of CD3B450.

[0800] Table 13. IC50 and K of anti-CD3 bivalent and monovalent CD3B376 and CD3B450 constructs d Affinity values

[0801]

[0802] Conformational stability of anti-CD3 monoclonal antibodies 1-10

[0803] The conformational stabilities of anti-CD3 antibodies CD3B376, CD3B389 (IgG1σ isotype of CD3B376; heavy chain is SEQ ID NO:729; light chain is SEQ ID NO:676), CD3B450 and CD3B467 (IgG1σ isotype of CD3B450; heavy chain is SEQ ID NO:728; light chain is SEQ ID NO:677) were determined by differential scanning calorimetry (DSC). The midpoint Tm of the thermal transition was determined from the thermal denaturation curves of each Ab candidate. Figures 12A to 12E Shows the thermal denaturation curves of anti-CD3 antibodies in PBS. Table 14 includes a summary of the Tm and enthalpy values (ΔH) of the thermal unfolding of anti-CD3 antibodies determined by DSC.

[0804] The DSC results indicate that all anti-CD3 antibodies CD3B376, CD3B389, CD3B450 and CD3B467 have folded domains. Based on the onset of unfolding, the relative stabilities of each antibody are as follows: CD3B389 < CD3B467 < CD3B376 < CD3B450. The anti-CD3 molecules tested by DSC showed some differences in thermal stability. The CD3B376 (IgG4 PAA) molecule showed three partially unresolved transitions at 59.7 °C, 62.4 °C and 69.2 °C, with a total unfolding enthalpy of 417.6 kcal / mol, while the CD3B450 (IgG4 PAA) molecule showed two unresolved transitions at 62.5 °C and 66.3 °C, with a total unfolding enthalpy of 545.1 kcal / mol. The CD3B389 (IgG1σ) molecule showed four transitions at 54.6 °C, 58.2 °C, 73.1 °C and 77.1 °C, with a total unfolding enthalpy of 401.7 kcal / mol, while the CD3B467 (IgG1σ) molecule showed four transitions at 56.3 °C, 59.6 °C, 66.5 °C and 75.6 °C, with a total unfolding enthalpy of 406.2 kcal / mol.

[0805] Table 14. Summary of thermal transition data of anti-CD3 antibodies in PBS. Values represent the average of repeated runs. HC peptide and LC peptide (SEQ ID NO in parentheses) sequence information is provided.

[0806]

[0807] The two IgG1 antibodies showed lower stability compared to the corresponding IgG4 PAA antibodies (comparing molecules with the same variable domains), with the Tm of the first transition being 5 °C - 6 °C lower ( Figure 13A and Figure 13B and Table 14).

[0808] Crystal structure of CD3B334 Fab complexed with CD3e N-terminal peptide 1-11

[0809] The anti-CD3 mAb CD3B334 (CD3H231 / CD3L137) was modified to increase the "humanity" index by replacing multiple framework residues in the antibody with human germline residues. This procedure generated an antibody CD3B334 with the following mutations: D43G / L49M / L50I / S62N / Q85E / H89Y in VL when compared to the parental VL CD3L124; and R10G / R13K / V73I / R79K / T83S / L96V in VH when compared to the parental VH CD3H219. The His-tagged Fab fragment of CD3B334 was expressed in HEK 293 Expi cells and purified using affinity chromatography and size exclusion chromatography. The N-terminal nonamer peptide of human CD3e was synthesized at New England Peptide (lot number V1108-19 / 21) and mixed with the Fab at a molar ratio of 10:1 (excess peptide). The complex was crystallized from a solution of 0.1 M Tris (pH 8.5) containing 4 M sodium formate by the vapor diffusion method. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of There is one complex molecule in the asymmetric unit. Using the crystal structure of the Fab as a search model, the structure of the complex was determined by molecular replacement at resolution.

[0810] CD3B334 binds to residues 1-6 of CD3e. The N-terminal Gln of the peptide is in the pyroglutamate form and is located between F107 of HCDR3 and L99 of LCDR3 in a hydrophobic environment. Two arginine residues, R52 and R56, from HCDR2 make electrostatic interactions with acidic residues of CD3. A total of 16 residues form the CD3B334 paratope. Residues from all CDRs except LCDR2 make direct contact with the CD3 peptide (within distance) (see Figure 18 ).

[0811] 2PSMA antibody

[0812] Generation of 2-1 PSMA cell line

[0813] Generate expression vectors presenting full-length chimpanzee PSMA (H2Q3K5_PANTR, SEQ ID NO:49) or full-length cynomolgus monkey PSMA (EHH56646.1, SEQ ID NO:50) to be used as screening tools to evaluate anti-PSMA leads using standard molecular biology techniques and an internal expression vector with a CMV promoter. Transiently transfect the vectors into suspended HEK293F cells using standard methods. Seed the transfected 293F suspended cells in serum-supplemented growth medium to become adherent and select for stable plasmid integration. Select single cell populations by serial dilution and quantify PSMA surface receptor expression by FACS using a rabbit polyclonal antibody affinity purified with (PSMAL antibody (Center) (catalog number OAAB02483, Aviva Systems Biology) as the primary antibody, while using an R-PE anti-rabbit secondary antibody (catalog number 111-116-144, Jackson ImmunoResearch Laboratories, Inc.) and rabbit polyclonal IgG (catalog number SC-532, Santa Cruz Biotechnology) as isotype controls).

[0814] Generate a human PSMA-expressing cell line using a lentivirus (Genecopoeia, catalog number EX-G0050-Lv105-10) containing full-length human PSMA (FOLH1_HUMAN, SEQ ID NO:51) and puromycin for the selection of PSMA-positive cells. Transduce PSMA-negative HEK293F cells (ATCC) with lentiviral particles to overexpress human PSMA. After transduction, select cells that are positive for PSMA and the resistance marker by treating the pooled cells to grow them in DMEM + 10% HI FBS (Life Technologies) supplemented with different concentrations of puromycin (Life Technologies).

[0815] In addition to the cell lines generated from HEK, several commercial cell lines were used for phage panning and binding as well as cytotoxicity assays. LNCaP clone FGC cells (ATCC, catalog number CRL-1740) are a commercially available human prostate cancer cell line. C4-2B cells were originally developed at MD Anderson and are derived from LNCaP FGC that grew and metastasized to the bone marrow in vivo (Thalmann et al., 1994, Cancer Research, Vol. 54, pp. 2577-2581).

[0816] Generation of soluble PSMA ECD protein 2-2

[0817] Recombinant chimpanzee PSMA extracellular domain (ECD) protein (chimpanzee PSMA ECD, SEQ ID NO:52) was generated for panning, and anti-PSMA leads were evaluated using standard molecular biology techniques and an internal expression vector with a CMV promoter. A chimpanzee PSMA ECD gene fragment (amino acids 44–750 of SEQ ID NO:49) with an N-terminal signal sequence (SEQ ID NO:594), an N-terminal Avitag (SEQ ID NO:595), and a 6-His tag (SEQ ID NO:596) was cloned using standard molecular biology techniques and an internal expression vector with a CMV promoter and transiently expressed in 293Expi cells (Invitrogen). cDNA was prepared using gene synthesis technology (U.S. Patent 6,670,127; U.S. Patent 6,521,427). The supernatant was collected by centrifugation and clarified. The protein was purified using a two-step purification process: 1) IMAC purification using a HisTrap HP column (GE Healthcare), and 2) size exclusion purification (Superdex 200, GeHealthcare), where the elution buffer was Dulbecco's phosphate-buffered saline containing 0.5 mM ZnCl 2 along with calcium and magnesium (Thermofisher, #14040) to stabilize PSMA dimerization. Fractions containing the protein of interest were pooled, and the protein concentration was determined by A280. This material was used for binding and affinity measurements and was designated PSMG8.

[0818] Chimpanzee PSMA ECD was also biotinylated for panning. A BirA plasmid that was co-transfected into mammalian cells to biotinylate proteins containing an Avi tag was generated internally. The BirA coding region (SEQ ID NO:597) was fused with a signal peptide from the mouse IgG heavy chain (SEQ ID NO:598), and an ER retention signal (KDEL (SEQ ID NO:716)) was added to the C-terminus to generate the BirA plasmid (SEQ ID NO:599). The constructed gene was cloned into an expression vector under the control of the CMV promoter. To generate biotinylated PSMA antigen, PSMA plasmid DNA was added to the transfection mixture at a 4-fold excess (w / w) over the BirA plasmid.

[0819] Biotinylation of chimpanzee PSMA ECD protein was performed via the Avi tag and by co-transfection of the BirA expression construct, and the resulting secreted protein was purified using a two-step purification process: 1) IMAC purification using a HisTrap HP column (GE Healthcare), and 2) size exclusion purification (Superdex 200, Ge Healthcare), where the elution buffer was Dulbecco's phosphate buffered saline (Thermofisher, #14040) containing 0.5 mM ZnCl2 and calcium and magnesium to stabilize PSMA dimerization. The endotoxin of the protein was tested before use in phage panning studies.

[0820] The recombinant cynomolgus monkey PSMA extracellular domain (ECD) protein (cynomolgus monkey PSMA ECD, SEQ ID NO:53) was cloned and expressed as previously described for chimpanzee PSMA ECD, which corresponds to amino acids 44 - 750 of SEQ ID NO:50 and has an N-terminal signal tag (SEQ ID NO:594), an N-terminal Avi tag (SEQ ID NO:595) and a 6His tag (SEQ ID NO:596). Biotinylation of cynomolgus monkey PSMA ECD protein was performed via the Avi tag and by co-transfection of the BirA expression construct, and the resulting secreted protein was purified using a two-step purification process with an IMAC HisTrap HP column (GE Healthcare) and a MonoAvidin column. The endotoxin of the protein was tested before use in phage panning studies. This material was also used for binding and affinity measurements and was designated PSMG1.

[0821] A second recombinant cynomolgus monkey PSMA ECD protein with IgG1 Fc (SEQ ID NO:593) (cynomolgus monkey PSMA Fc, SEQ ID NO:54) was cloned and expressed using standard molecular biology techniques and an internal expression vector with a CMV promoter. The cynomolgus monkey PSMA Fc protein was transiently expressed in 293HEK-expi cells. Five days after transfection, the transient transfection of PSMG3 in HEK293 Expi cells was harvested, clarified by centrifugation (30 min, 6000 rpm) and filtered (0.2 μ PES membrane, Corning). The relative amount of IgG was determined by an Octet instrument (ForteBio) and using purified known IgG (same isotype) incorporated into the used medium to generate a standard curve.

[0822] The clarified cynomolgus PSMA Fc supernatant was loaded onto an equilibrated (dPBS, pH 7.2) HiTrap MabSelect Sure protein A column (GE Healthcare) at a relative concentration of approximately 30 mg protein / ml resin. After loading, the column was washed with dPBS (pH 7.2) and the protein was eluted with 10 column volumes of 0.1 M sodium acetate (pH 3.5). The peak fractions were pooled, neutralized with 2 M Tris (pH 7), and filtered (0.2 μ). The neutralized protein sample was dialyzed overnight at 4 °C against three changes of dPBS containing Ca2+, Mg2+ and 0.5 mM ZnCl2 (pH 7.2). The next day, the sample was removed from the dialysis buffer, filtered (0.2 μ), and the protein concentration was determined by absorbance at 280 nm on a BioTek SynergyHTTM spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (Dionex HPLC system). The endotoxin level was measured using the LAL assay (Pyrotell-T, Associates of Cape Cod). The purified protein was stored at 4 °C.

[0823] The recombinant human PSMA extracellular domain (ECD) protein (human PSMA ECD, SEQ ID NO:55) was cloned, expressed, and purified as previously described for chimpanzee and cynomolgus PSMA ECD proteins, corresponding to amino acids 44 - 750 of SEQ ID NO:51 and having an N-terminal Avi tag and a 6His tag (SEQ ID NO:596).

[0824] Identification of anti-chimpanzee and anti-human PSMA Fab 2-3

[0825] Panning with recombinant protein

[0826] First solution panning of a de novo human Fab-pIX library consisting of VH1-69, 3-23, and 5-51 heavy chain libraries paired with a library of four human VL germline genes (A27, B3, L6, O12) [Shi, L. et al., J Mol Biol, 2010, Vol. 397, No. 2: pp. 385-396, WO 2009 / 085462] was performed using an alternating panning method in which one round of phage capture was performed on streptavidin magnetic beads (Invitrogen, catalog number 112.05D, lot number 62992920) coated with biotinylated chimpanzee PSMA ECD according to the manufacturer's protocol, followed by phage capture on ProtG beads (Invitrogen, catalog number 10003D) coated with cynomolgus monkey PSMA-Fc according to the manufacturer's protocol, and then phage capture on Sera-mag Double Speed neutravidin magnetic beads (Thermo, catalog number 7815-2104-011150) coated with biotinylated chimpanzee PSMA ECD according to the manufacturer's protocol. This panning yielded two hits: PSMM18 and PSMM25.

[0827] Whole cell panning of anti-PSMA Fab

[0828] Additional panning experiments on whole cells were performed using the output from the first round of the chimpanzee ECD panning experiment described above or a fresh de novo phage library as input. Briefly, phage were generated by helper phage infection and concentrated by PEG / NaCl precipitation according to standard protocols known in the art. The phage library was pre-cleared by gently shaking overnight at 4°C on untransfected parental HEK293F cells. After PEG / NaCl precipitation, the pre-cleared library was incubated with HEK293 cells or LNCAP cells expressing chimpanzee PSMA while gently shaking at 4°C for 2 hours. Removal of unbound phage and recovery of phage-bound cells were performed by Ficoll gradient, and after several washing steps, the cells carrying the bound phage were incubated with 1 mL of TG-1 Escherichia coli (E. coli) culture at 37°C for 30 minutes without agitation. The resulting mixture was plated on LB-carbenicillin-1% glucose plates and grown overnight at 37°C. The process was then repeated for subsequent rounds of panning.

[0829] Conversion of phage Fab-pIX into Fab-His to generate E. coli supernatant

[0830] The resulting phage Fab-pIX hits were converted to Fab-His using standard procedures. Plasmid DNA was isolated from E. coli (Plasmid Plus Maxi kit, Qiagen catalog number 12963) from phage panning and subjected to NheI / SpeI restriction digestion. The resulting 5400 bp and 100 bp fragments were separated on a 0.8% agarose gel and the 5400 bp fragment was gel purified (MinElute PCR purification kit, Qiagen catalog number 28006). The purified 5400 bp band was self-ligated using T4 ligase and the resulting product (encoding the Fab-his fusion) was transformed back into the TG-1 E. coli strain and cloned separately. Fab-His supernatant was produced from the clones by inducing the cultures overnight with 1 mM IPTG. After centrifuging the overnight cultures, the clarified supernatant was prepared for use in downstream assays. To determine the relative expression levels of different Fab-his supernatants, anti-k (Southern Biotech, catalog number 2061-05) ELISA was performed on serial dilutions of the supernatants. All clones tested showed similar Fab-his expression (data not shown).

[0831] Cell binding of Fab-his fusion from E. coli

[0832] Cell-based binding assays were designed to evaluate the binding ability of individual Fab-his fusions from E. coli supernatants to cells expressing PSMA. After pIX excision, individual Fab clones were isolated from the output of the 3rd round of all panning experiments. The binding of Fab clones to HEK cells expressing cynomolgus and chimpanzee PSMA, as well as to human PSMA on LNCaP cells, was tested. Briefly, cells expressing PSMA were aliquoted at a density of 200,000 / well into V-bottom plates (CoStar 3357) and incubated with (100 μl) supernatant expressing the Fab fragment for 1 hour on ice. The cells were washed twice with PBS containing 2% FBS and stained with mouse anti-human κ-RPE antibody (Life Technologies, catalog number MH10514) for 1 hour on ice. The cells were washed twice with PBS containing 2% FBS and resuspended in 100 μL of the same wash buffer. The plates were read on a BD FACSArray flow cytometer. FACS data were analyzed in FlowJo software by live gating of the healthy cell population using forward scatter and side scatter, and then analyzing the PE staining of the cells within that gate. The mean fluorescence intensity (MFI) was calculated and exported to Microsoft Excel. Fab clones that showed binding ≥3-fold background for all three PSMA species (cynomolgus, chimpanzee, and human) and did not show binding to the HEK293 cell line were labeled "preliminary positive". The Fab was sequenced and moved forward to be cloned into a mammalian expression vector for rescreening. True positives were selected from the binding of Fab supernatants expressed from mammalian cells to cell lines expressing PSMA.

[0833] Preparation of mammalian Fab

[0834] To convert E. coli Fabs to mammalian-expressed Fabs, In-FusionHD cloning (ClonTech catalog number 638918) was utilized according to the manufacturer's protocol. Briefly, the nucleotide sequences of clones that had passed preliminary screening and been moved into mammalian Fab format were loaded into the "InFu Primer Finder v1.2.3" program (internally developed software), which generated a list of isotype-specific PCR primers for generating PCR fragments for seamless cloning into the huKappa_muIgGSP and huG1 Fab expression vectors. These vectors are internal vectors with a CMV promoter based on pcDNA3.1. After the seamless cloning process, E. coli clones were isolated using standard protocols, sequence-verified, and transfected into HEK293 cells. Mammalian PSMA Fabs for confirmation of binding to cell lines expressing PSMA were prepared by harvesting 20 ml supernatant from the transfection after 5 days.

[0835] Rescreen hits from whole cell panning in mammalian supernatant

[0836] Validation of mammalian expressed Fab supernatants was performed using the whole cell binding assay described above. Fabs were tested for binding to human PSMA (LNCaP), chimpanzee and cynomolgus monkey cells, and counter-screened for non-binding to the parental HEK cell line. Table 19 shows the hit characteristics of mammalian Fab supernatants that bind to cells expressing PSMA. Many hits from E. coli supernatants could not be confirmed by mammalian expressed proteins. PSMM48 showed high binding to cells expressing cynomolgus PSMA and some binding to cells expressing chimpanzee PSMA, but not to LNCaP cells expressing human PSMA. PSMM56 showed similar characteristics, but some binding to LNCaP cells. PSMM69–80 bound to LNCaP cells, but not to cells expressing chimpanzee PSMA or cynomolgus PSMA. Mammalian Fab supernatants PSMM52, M56 and M57 bound to all three cell lines. PSMM50, M51 and M54 showed more binding to chimpanzee or cynomolgus monkeys. M58 showed slight chimpanzee and cynomolgus monkey binding.

[0837] Table 19. Characteristics of hits of mammalian Fabs binding to cells expressing PSMA measured by Geo-MFI (Geometric Mean Fluorescence Intensity) Protein

[0838]

[0839]

[0840]

[0841]

[0842] Dose response curves of mammalian-expressed Fabs

[0843] Once mammalian expressed Fab clones were confirmed to bind positively to PSMA expressing cell lines as pure Fab supernatants, supernatants were normalized for protein concentration by Octet or protein gel and dose response curves were completed using the previously described protocol to confirm PSMA binding. Figures 22 to 24 show titration curves for hits that showed binding to all three PSMA expressing cells. Figure 22A , Figure 22B , Figure 22C and Figure 22D Titration curves of anti-PSMA panning hits against LNCaP cells are shown. Figure 23A , Figure 23B , Figure 23C and Figure 23DShows the titration curve of anti-PSMA panning hits against chimpanzee PSMA HEK cells. Figure 24A , Figure 24B , Figure 24C and Figure 24D Show the titration curve of anti-PSMA panning hits against cynomolgus monkey PSMA HEK cells. The binding characteristics of the hits were compared between cell lines expressing PSMA of different species. PSMM52 supernatant was used as a positive control throughout the experiment. Due to the N-linked glycosylation sites in the CDR, the binding to PSMA-negative parental HEK cell lines or the lack of binding to PSMA-positive cell lines reduced the priority of some hits. Eleven Fab hits remained, and ten hits were cloned into a human IgG4-PAA heavy chain construct and used to generate PSMA×CD3 bispecific antibodies. These hits showed cross-species binding within 3-fold of each other and were transferred into the bispecific antibody format to test T cell redirected killing of PSMA-positive targets. The panning antigen for each hit is shown in Table 20.

[0844] Table 20. Antigens of each panning hit

[0845]

[0846] Preparation of anti-PSMA mAb

[0847] A total of 12 clones that showed binding to all three PSMA-expressing cells were finally converted into mAb IgG4 with Fc replacement S228P, F234A, and L235A (PAA) isotype by restriction cloning. Briefly, the construct corresponding to the Fab clone that had passed the initial screening was digested with HindIII and ApaI. The gel-purified fragment was ligated into an expression vector with CMV promoter vDR000215, and the CMV-driven expression vector contained human IgG4-PAAFc for full mAb expression. This allowed for the rapid generation of bispecific antibodies. The expression vectors previously described were used to express the heavy and light chains of each PSMA mAb, and the two vectors were transiently co-transfected into 293Expi or CHO cell lines for mAb expression. The CDR sequences of the cross-species positive PSMA Fab generated by phage panning are shown in Table 21 below. The VH sequences and VL sequences of the selected Fab are shown in Table 22 below. The heavy chain sequences and light chain sequences of the mAb generated from the Fab are shown in Table 23 below.

[0848] Table 21. CDR sequences of Fab from phage panning (defined according to Kabat) (corresponding SEQ ID NOs are listed in parentheses)

[0849]

[0850]

[0851]

[0852] Table 22. VH and VL sequences of PSMA Fab

[0853]

[0854]

[0855]

[0856] Table 23. Heavy and light chain sequences of PSMA monoclonal antibodies with corresponding SEQ ID NOs

[0857]

[0858]

[0859]

[0860]

[0861]

[0862]

[0863]

[0864] The monospecific anti-PSMA antibody PSMB119 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:130 and VL with SEQ ID NO:131, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB120 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:128 and VL with SEQ ID NO:129, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB121 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:126 and VL with SEQ ID NO:127, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB122 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:125 and VL with SEQ ID NO:111, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB123 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:123 and VL with SEQ ID NO:124, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB124 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:121 and VL with SEQ ID NO:122, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB126 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:118 and VL with SEQ ID NO:119, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB127 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:116 and VL with SEQ ID NO:117, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB128 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:114 and VL with SEQ ID NO:115, and an IgG4 constant region having S228P, F234A, and L235A substitutions.The monospecific anti-PSMA antibody PSMB129 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:110 and VL with SEQ ID NO:111, and an IgG4 constant region having S228P, F234A, and L235A substitutions. The monospecific anti-PSMA antibody PSMB130 was generated, which comprises a VH region and a VL region having VH with SEQ ID NO:112 and VL with SEQ ID NO:113, and an IgG4 constant region having S228P, F234A, and L235A substitutions.

[0865] Crystal structures of human PSMA ECD bound to anti-PSMA Fab PSMB83 (also known as PSMM84) 2-5

[0866] PSMA is a homodimeric protein expressed on the cell surface. PSMA is a type II integral glycoprotein of 750 residues per monomer, consisting of a large ECD domain (705 residues) with peptidase activity, a single-pass TM domain, and a short 19-residue intracellular domain. The crystal structure of the extracellular region (ECD) of human PSMA bound to the anti-PSMA Fab arm of the bispecific antibody PS3B27 was determined at

[0867] The extracellular region (residues 44 - 750) of human PSMA was expressed in High Five TM insect cells with an N-terminal gp67 signal peptide and a subsequent cleavable hexahistidine tag (SEQ ID NO:596). The secreted protein was purified from the supernatant by a three-step procedure that included: initial Ni 2+ -NTA affinity capture, TEV-mediated histidine tag cleavage, followed by a reverse affinity chromatography step and finally a size exclusion chromatography step. The purified PSMA-ECD was flash-frozen in liquid nitrogen and stored at -80 °C in 10 mM HEPES (pH 7.4, 150 mM NaCl, 2 mM CaCl 2 , 0.1 mM ZnCl 2 ).

[0868] The Fab of PSMB83 (also known as PSMM84), which is the parental anti-PSMA Fab arm in the bispecific antibody PS3B27, was expressed in HEK293Expi cells with a hexahistidine tag (SEQ ID NO:596) and purified using affinity chromatography (HisTrap, GE Healthcare) and size exclusion chromatography (SEC-300, Phenomenex Yarra). The Fab was stored at 4 °C in 50 mM NaCl, 20 mM Tris (pH 7.4).

[0869] The human PSMA ECD / PSMB83 Fab complex was prepared by a three-step procedure. First, the Fab was buffer-exchanged into 20 mM MES (pH 6.0, 150 mM NaCl). Then, the Fab and PSMA were mixed (1.5 molar excess of Fab relative to PSMA monomer) and incubated overnight at 4 °C while dialyzing into 20 mM MES at pH 6.0. Finally, the complex was bound to a MonoS 5 / 50 column in 20 mM MES (pH 6.0) and eluted with an NaCl gradient.

[0870] Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapor diffusion method and a Mosquito LCP robot (TTP Labtech) at 20 °C. Crystals of PSMB83 Fab bound to human PSMA ECD were grown from 18% PEG 3 kDa, 0.2 M (NH 4 ) 2 SO 4 , 0.1 M Tris (pH 8.5) and using microseeding and a PSMA / Fab complex that was initially 7.3 mg / mL. Crystals of free PSMB83 Fab were obtained from 25% PEG 3 kDa, 0.2 M LiCl, 0.1 M acetate (pH 4.5), where the Fab was initially 8.8 mg / mL.

[0871] The structure was solved by molecular replacement (MR) using Phaser (Phaser crystallography software, University of Cambridge). The MR search model for the PSMB83 (also known as PSMM84) Fab structure was the PDB code 4M6O. The PSMA (PDB code: 2C6G) and PSMB83 Fab ( structure at the resolution; data not shown) were used as the MR search models to solve the PSMA / Fab complex structure. The structure was refined using PHENIX (Adams et al., 2004) and model building was performed using COOT (Emsley and Cowtan, 2004). All other crystallographic calculations were performed using the CCP4 suite of programs (Collaborative Computational Project Number 4, 1994). All molecular graphics were generated using PyMol (PyMOL Molecular Graphics System, version 1.4.1, LLC.) and the complementarity-determining regions (CDRs) were determined using the Kabat definition.

[0872] The PSMA / Fab structure includes Fab light chain residues 1 - 211, Fab heavy chain residues 1 - 224 (except for disordered residues 138 - 146), and PSMA residues 56 - 750, which correspond to the protease (residues 56 - 116 and 352 - 590), apical (residues 117 - 351), and helical (residues 591 - 750) domains, as well as seven of the ten possible N - linked glycans (Asn - 76, - 121, - 140, - 195, - 459, - 476, and - 638) per PSMA dimer subunit. The PSMA active site is located at the interface between these three domains and it contains two zinc atoms coordinated by histidine (H377 and H553) and glutamate / aspartate (D387, catalytic E424, E425, and D453) residues and water molecules. The crystal asymmetric unit contains one PSMA dimer, in which each subunit binds in a manner similar to PSMB83Fab. The Fab / PSMA binding site is precisely defined by an electron density map that allows reliable localization of the binding residues. The Fab and PSMA molecules are numbered in sequence in Figures 25 to 30 in sequence.

[0873] PSMB83 epitope, paratope, and interactions. PSMB83 is the parental anti - PSMA Fab arm in the bispecific antibody PS3B27, which recognizes conformational and discontinuous epitopes in the apical domain of PSMA ( Figure 25 ). The surface area of PSMA buried by the Fab is approximately Specifically, the PSMB83 epitope residues are I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324 - P326. Helix α7 (residues Y299 - E307) is a general region of the epitope and binds to the Fab heavy and light chain CDRs. At one end of the helix, Y299 and Y300 form an aromatic cluster with Fab residues Y57 H , W94 L and PSMA residues F235 and P237, while at the other end of the helix, E307 forms a salt bridge with R91 L and hydrogen bond Y32 L . Figure 26 and Figure 27 show the major interactions of PSMA with the light and heavy chains of PSMB83. The PSMB83 epitope residues are conserved between humans and cynomolgus monkeys ( Figure 28 ), and the bispecific antibody PS3B27 has been shown to bind to human and cynomolgus monkey PSMA with similar affinities. In contrast, the human - to - mouse G238A mutation, especially the Y300D epitope mutation, is expected to reduce the binding affinity of PSMB83 to mouse PSMA compared to humans. The Y300D mutation disrupts the binding to N59H hydrogen bond contacts and with W94 L π - stacking interactions.

[0874] The PSMB83 complementary site is composed of residues in all CDRs except CDR - L2 and CDR - H1( Figure 29 ). Specifically, the complementary site residues are light - chain S30 L , Y32 L , R91 L , S92 L , W94 L and heavy - chain G56 H - N59 H , K65 H , G66 H , Y101 H , V107 H and D109 H . Figure 30 shows the interaction contacts between PSMA and PSMB83. The accessible positions of the epitope facilitate the binding of the PSMB83 Fab arm to membrane - bound PSMA in the PS3B27 bispecific antibody, while the other Fab arm remains bound to CD3 in the T - cell membrane. PSMB83 is not expected to inhibit PSMA enzyme activity because the antibody binds away from the active site and does not cause any significant structural changes in PSMA that could affect enzyme function, such as loop movements closing the active site or replacement of catalytic residues (for Cα superposition of PSMA molecules in the bound - Fab and unbound - Fab structures, the RMSD is (Barinka et al., 2007)).

[0875] 2-6 Anti-PSMA affinity maturation

[0876] The anti-PSMA Fab phage clones from two PSMA affinity maturation libraries were affinity matured to identify antibodies with increased binding affinity compared to the parental PSMB127 (fab ID = PSMB83, also known as PSMM84). Two libraries were generated for the affinity maturation of PSMB127. In the first library, the heavy chain CDR1 and CDR2 were randomized according to the design in Table 24 (PH9H9L1). The H-CDR3 fragment was PCR amplified from pDR000024032 and digested with SacII + XhoI. This fragment was cloned into the PH9H9L1 / PH9L3 library. It was transformed into Escherichia coli MC1061F' cells, and phage displaying this Fab library was generated. In the second library, the light chain CDR was randomized according to the design in Table 25 (PH9L3L3). The heavy chain from PSMB83 (PSMH360) was PCR amplified and digested with NcoI + XhoI. This fragment was cloned into the PH9L3L3 library DNA (ELN: de novo 2010 phage library SRI-021). It was transformed into Escherichia coli MC1061F' cells, and phage displaying this Fab library was generated.

[0877] Table 24: PH9H9L1 library design

[0878] Position Parent AA Library AA 30 S D, K, S 31 S D, N, S, T 32 Yes A, D, S, Y 33 A A, D, G, S, W, Y 35 S H, N, S 50 A A, E, L, N, R, T, W, Y 52 S A, D, L, N, R, S 54 S A, E, N, S, Y 57 S D, N, R, S, T, Y 59 Yes E, G, N, Q, R, Y

[0879] Table 25: PH9L3L3 library design

[0880]

[0881]

[0882] Solution panning of the PSMA affinity maturation Fab-pIX library against biotinylated human PSMA ECD was performed for three rounds. The phage-bound antigen was captured on neutravidin magnetic beads (GE Healthcare Life Science, catalog number 78152104011150) according to the manufacturer's protocol, then washed thoroughly in 1x PBST (0.05% tween 20), and incubated for a long time with unlabeled PSMA ECD in 500-fold molar excess of biotinylated antigen. This panning yielded the clones PSMXP46R3_59H09, PSMXP46R3_59H06, PSMXP46R3_59E03, PSMXP46R3_59C09, PSMXP46R3_59H01, PSMXP46R3_59F11, and PSMXP46R3_59F07.

[0883] To determine the expression level of anti-PSMA fab clones, 96-well Maxisorb plates were coated overnight at 4 °C with anti-human Fd IgG, washed, and blocked for 1 h with 3% milk-PBS-0.05% Tween. Phage supernatant samples were serially diluted 11 times in blocking buffer, 2-fold dilutions, with the final well being blank. 100 μl of these solutions were captured on the coated plates for 1 h. The plates were washed, 100 μl of anti-F(ab’)2-HRP antibody was added and incubated for 1 h. The plates were washed and developed with 100 μl of peroxidase reagent, and luminescence was read on Envision( Figure 31 ).

[0884] To determine the binding of anti-PSMA fab clones to human and cynomolgus monkey recombinant proteins, 96-well Maxisorb plates were coated overnight at 4 °C with 100 μl of 5 μg / ml neutravidin. The plates were washed and blocked for 1 h with 3% milk-PBS-0.05% Tween. Recombinant biotinylated human and cynomolgus monkey PSMA proteins were captured at 2.5 μg / ml for 1 h at room temperature. The plates were washed, and 100 μl of 2-fold serial dilutions of the fab supernatant were captured for 1 h at RT. The plates were washed, then incubated with 200 μl of PBST containing 0.3% milk for 2.5 h to wash away some of the weakly binding fabs. Then incubated again with fresh 200 μl of PBST containing 0.3% milk for 30 min to remove even weaker binding fabs. The plates were washed, 100 μl of anti-F(ab’)2-HRP antibody was added and incubated for 1 h. The plates were washed and developed with 100 μl of peroxidase reagent, and luminescence was read on Envision( Figure 32 and Figure 33 ).

[0885] Figure 31 Protein expression of the parental Fab and affinity matured Fab was shown to be similar. The y-axis values represent the luminescence of the detection reagent, which is equal to the abundance of the fab protein on the dilution curve; the higher the luminescence reading, the more protein in the well, and the protein decreases after successive two-fold dilutions. There was more protein in the wells with affinity matured fabs, but the increase compared to the parental was at most five-fold as shown by the EC50 value (which is the protein concentration that provides half of the maximum response). These data indicate that Figure 32 and Figure 33 the differences in PSMA binding characteristics in

[0886] Figure 32Indicates improved binding of the affinity matured Fab to the human recombinant antigen compared to the parental anti-PSMA Fab (PSMB83). Similarly, the y-axis of the graph represents the luminescence value. In this case, the larger the value, the more Fab bound to the human PSMA protein. This is a measure of binding as increasing Fab concentration (along the x-axis) results in a higher luminescence value. The binding of the parental Fab under these conditions is negligible, as confirmed by the absence of signal even at high concentrations (open circles along the x-axis). Binding of the affinity matured fab was observed at the concentrations tested, which is equivalent to a stronger binding ability to the human PSMA protein. Given that the binding of the parental Fab to the human PSMA protein is zero, no EC50 is generated.

[0887] Figure 33 Indicates improved binding of the affinity matured Fab to the cynomolgus monkey recombinant antigen compared to the parental anti-PSMA Fab (PSMB83). Similarly, the y-axis of the graph represents the luminescence value. In this case, the larger the value, the more Fab bound to the cynomolgus monkey PSMA protein. This is a measure of binding as increasing Fab concentration (along the x-axis) results in a higher luminescence value. The binding of the parental Fab under these conditions is negligible, as confirmed by the absence of signal even at high concentrations (open circles along the x-axis). Binding of the affinity matured fab was observed at the concentrations tested, which is equivalent to a stronger binding ability to the human PSMA protein. Given that the binding of the parental Fab to the human PSMA protein is zero, no EC50 is generated for direct comparison.

[0888] Overall, the Fab binding characteristics of the phage indicate improved binding to both human and cynomolgus monkey recombinant antigens compared to the parental anti-PSMA mAb (PSMB127). This improvement is not the result of differences in Fab expression characteristics, as Figure 33 and Figure 34 shown, depicting the binding of the affinity matured Fab normalized to the Fab expression level. The top five Fab candidates identified from the ELISA screen were generated as monoclonal antibodies on IgG4 PAA. Table 26 lists the subsequent Mab identifiers, and Tables 27 and 28 describe the sequences of their variable regions and the sequences of the heavy and light chains, respectively.

[0889] Table 26: Top five affinity matured antibodies identified by ELISA

[0890]

[0891] Table 27. VH and VL sequences of the top five PSMA Fab candidates

[0892]

[0893]

[0894] Table 28. Heavy and light chain sequences of the top five PSMA candidates in monoclonal antibody form on IgG4PAA

[0895]

[0896]

[0897]

[0898] Three different HCs and four different LCs were combined in a matrix form to expand the diversity of hits (Table 29). Considering that the methionine in the CDR2 of PSMH860 is a post-translational risk, a new sequence was generated with M64L and identified as PSMH865. PSMH865 was paired with PSML160 to generate Mab PSMB365.

[0899] Table 29: Matrix form of 3 heavy chains and 4 light chains combined

[0900] PSMH859 PSMH860 PSMH862 PH9L3 PSMB344 PSMB347 PSMB358 PSML158 -- PSMB361 PSMB349 PSML159 PSMB345 PSMB362 PSMB359 PSML160 PSMB346 PSMB363 PSMB360

[0901] Table 30 and Table 31 show the sequences of the matrix-recombined variable regions and the sequences of the heavy and light chains, respectively.

[0902] Table 30. VH and VL sequences of matrix-recombined PSMA hits

[0903]

[0904]

[0905] Table 31. Heavy and light chain sequences of matrix-recombined PSMA hits

[0906]

[0907]

[0908]

[0909]

[0910] Table 32 shows the CDR sequences of all affinity-matured PSMA hits.

[0911] Table 32. CDR sequences of affinity matured PSMA hits

[0912]

[0913]

[0914] Preparation and functional evaluation of PSMA×CD3 bispecific antibodies

[0915] 3-1 Generation of PSMA×CD3 bispecific antibodies

[0916] Two types of affinity matured PSMA×CD3 bispecific antibodies were generated: one specific for the targeting arm (e.g., affinity matured anti-PSMA) which was recombined with a high affinity CD3 arm i.e., CD3B376 (VH SEQ ID NO:652, VL SEQ ID NO:661; HC SEQ ID NO:640, LC SEQ ID NO:676) CD3 arm or a low affinity CD3B450 (VH SEQ ID NO:657, VL SEQ ID NO:678, HC SEQ ID NO:675, LC SEQ ID NO:677) arm.

[0917] These parental mAbs are in the GenMab format (Labrijn et al., 2013), where the target parent (PSMA) contains the 409R GenMab mutation (the native amino acid of IgG4), while the killing parent (CD3) contains the F405L GenMab mutation and the R409K mutation. The monospecific anti-CD3 antibody is expressed as IgG4 and has Fc substitutions S228P, F234A, L235A, F405L and R409K (CD3 arm) in its Fc region (numbered according to the EU index). The targeting parent (PSMA) is on human IgG4 with Fc substitutions S228P, F234A, L235A. The monospecific antibodies are expressed in the HEK cell line under the CMV promoter.

[0918] The parental PSMA and CD3 antibodies were purified using a Protein A column with an elution buffer of 100 mM NaAc pH 3.5 and a neutralization buffer of 2.5 M Tris pH 7.2. The neutralized parental mAbs were used to prepare the PSMA×CD3 bispecific antibody. A portion of the parental mab was further buffer exchanged into D-PBS (pH 7.2) buffer for analytical measurements and assays.

[0919] After purification, controlled Fab arm exchange was performed to prepare the bispecific antibody. The parental PSMA antibody was mixed with the desired parental CD3 antibody in 75 mM of 2-MEA (2-mercaptoethylamine) under reducing conditions and incubated at 31 °C for 4 hours or overnight at room temperature. The recombination reaction was based on the molar ratio, where a 6% excess of the PSMA parental mAb was used to minimize the remaining CD3 parental mAb after recombination. The recombinant was then dialyzed against 1xDPBS (pH 7.2) to remove the reducing agent.

[0920] The final bispecific antibodies generated, along with the parental mAbs (i.e., PSMA, CD3 or blank) used in the recombination reaction, are listed in Table 33.

[0921] The selected PSMA hits were also paired with a non-cytotoxic arm (blank) to generate a negative control for testing purposes. For the control bispecific antibody B2M1, an RSV antibody in IgG4 PAA form (VH SEQ ID NO: 610, VL SEQ ID NO: 611) was generated and purified, and combined with the CD3 arm CD3B219-F405L, R409K to generate CD3B288 (CD3 × blank), or combined with the PSMA arms PSMB122, PSMB126, PSMB130 to generate PS3B37, PS3B39, and PS3B40 (PSMA × blank), respectively. These PSMA-specific affinity matured Mabs were hybridized with CD3B219 and CD3B376 (as described above) to generate the bispecific antibodies shown in Table 32.

[0922] Table 33. Generation of affinity matured PSMA×CD3 bispecific antibodies from affinity matured PSMA hits 3-2 Evaluation of affinity matured bispecific abs of PSMA×CD3 in LNCAP cell binding

[0923]

[0924]

[0925] Figures 14 to 16

[0926] The binding of PSMA×CD3 bispecific antibodies to the PSMA-positive cell line LNCAP and the PSMA-negative cell line PC3 was tested. To evaluate the binding ability of the PSMA bispecific antibodies, a cell binding assay (as described previously) was utilized. The bispecific antibodies were normalized against the protein concentration and then incubated with the same number of cells expressing human or cynomolgus monkey PSMA. The MFI at each concentration was collected by flow cytometry and plotted as a function of concentration. The data were transformed by log10 and then plotted. Nonlinear regression of the binding curves was performed to determine the EC50.

[0927] These relative values were used to rank the PSMA that bound to the target cells. Figure 16 The LNCAP binding of all the prepared bispecific antibodies is shown. In Figure 14 none of the constructs showed binding to the PSMA-negative cell line. In Figure 15 and ​ compared to the parental Mab PS2B27, all the affinity matured hits showed increased binding affinity and increased cMax as indicated by a left-shifted curve.

[0928] The interaction of affinity matured bispecific antibodies with recombinant cynomolgus PSMA ECD and human PSMA ECD was studied by surface plasmon resonance (SPR) using a ProteOn XPR36 system (BioRad), as previously described for recombinant chimpanzee PSMA ECD. All bispecific antibodies bound both targets with essentially the same affinity, with KD ranges of 0.05 nM to 0.27 nM for human PSMA ECD and 0.05 nM to 0.23 nM for cynomolgus PSMA ECD.

[0929] Evaluation of bispecific Abs with PSMA×CD3 affinity maturation in 3-3 functional cell killing assay

[0930] Based on the above data, affinity measurements, and sequence identity, the ability of three PSMA antibodies, PSMB347, PSMB360, and PSMB365, as bispecific antibodies with CD3B219 or CD3B376, to mediate PSMA-specific redirected T cell cytotoxicity was further characterized. T-cell mediated killing was measured using a caspase cytotoxicity assay that indirectly measures cell killing by cleavage of a fluorescent substrate by active caspase 3 / 7. Cleavage of the substrate produces a fluorescent DNA dye, where the fluorescence is confined to the cell nucleus. During the entire assay, repeated fluorescence measurements were made in each well using a 10X motorized objective capable of precisely imaging the wells at the same coordinates. The target cell population was identified based on defined size limits and / or by using a second marker. Cryopreserved Pan CD3+ T cells (purchased from Biological Specialty Corporation, Colmar, PA) were isolated from normal healthy donors by negative selection. Prostate cancer cells expressing PSMA (LNCaP, C42) were cultured in RPMI 1640 (purchased from Life Technologies) with 10% HI FBS + supplements.

[0931] In the absence of selection reagents, T cells and target cells were mixed at an effector-to-target ratio (E:T) of 3:1 in phenol red-free RPMI + 10% FBS and supplements (Life Technologies), and 0.6 μL of NucView caspase reagent (Essen Bioscience) was added to each mL of cells according to the manufacturer's instructions. A total volume of 0.1 mL of cells was added to the appropriate wells of a clear 96-well flat-bottom plate (BD Falcon). Bispecific antibodies PS3B27 (CD3×PSMA), CD3B288 (CD3×blank), or PS3B46 (PSMA×blank) were prepared at 2X final concentration in phenol red-free RPMI as described above, and 0.1 mL of the compound was added to each well. After incubating for 30 minutes at room temperature to minimize cell aggregation at the well edges, the plate was transferred to a Zoom Incucyte instrument (Essen Bioscience). The Incucyte instrument was located in a humidified incubator set at 37 °C and 5% CO2.

[0932] Treatment definitions on the Incucyte were designed for each cell line tested according to the manufacturing guidelines. Measurements were taken every six hours until a plateau in the caspase signal was observed, followed by three or more consecutive decreases starting from the maximum signal in the well containing the highest concentration of the test compound. As Figure 17 shown in the data, the curves of PS3B80, PS3B79, PS3B89, PS3B90, PS3B63, and PS3B72 were shifted to the left, indicating higher potency than PS3B27. The blank arm control did not induce cell death as expected.

[0933] 3-4 Antitumor efficacy of LnCaP xenografts in tumorigenesis prevention in humanized NSG mice

[0934] In male NOD.Cg-Prkdc scid Il2rg tm1WjlEvaluate the efficacy of PS3B79 and PS3B90 in established 3D LnCaP AR.TB human prostate cancer xenografts in / SzJ(NSG) mice. PS3B79 and PS3B90 were administered at 2.5 mg / kg and 5 mg / kg or the blank × CD3B376 antibody control at q3d-q4d on days 36, 39, 43, 47, 50, 53, 56, 60, and 63, for a total of 8 administrations. On day 53 after tumor implantation (which was the last date of the study), when nine (9) animals were retained in each group, tumor growth inhibition (%TGI) was calculated. The following cases of statistically significant tumor growth inhibition were observed compared to the blank × CD3 control: For PS3B79 at 5 mg / kg, the observed TGI was 42% (two-way ANOVA with Bonferroni test, *p<0.0001, Figure 20 ), and for PS3B90 at 2.5 mg / kg and 5 mg / kg, the observed TGIs were 53% and 33% respectively (two-way ANOVA with Bonferroni test, *p<0.001, Figure 21 ). Thus, CD3B376 is capable of inducing T cell activation and cytotoxicity in vivo and results in tumor growth inhibition in a bispecific form with high-affinity PSMA-binding arms PSMB360 and PSMB365.

[0935] Preparation and functional evaluation of 4IL1RAP×CD3 bispecific antibody

[0936] The IL1RAP monoclonal antibodies used in and suitable for use in the bispecific antibodies of the present disclosure are described in U.S. Patent Application Publication 20170121420A1, the disclosure of which is hereby incorporated by reference in its entirety. Fifteen monospecific IL1RAP antibodies (see Table 6 of US20170121420A1) were expressed as IgG4 with Fc substitutions S228P, L234A, and L235A. The CD3 parental mAb was expressed as IgG4 with S228P, L234A, L235A, F405L, and R409K (numbered according to EU index). The monospecific anti-CD3 antibody CD3B220 was also generated, which contains a VH region and a VL region with VH having SEQ ID NO:92 and VL having SEQ ID NO:93, and an IgG4 constant region with S228P, L234A, L235A, F405L, and R409K substitutions.

[0937] Purify the monospecific antibodies using standard methods and a protein A column. After elution, the pool was neutralized to pH 7 and dialyzed into 1x D-PBS at pH 7.2.

[0938] A bispecific IL1RAP×CD3 antibody is generated by controlled Fab-arm exchange of a monospecific CD3 mAb (CD3B376: VH of SEQ ID NO:652 and VL of SEQ ID NO:661; or CD3B450: VH of SEQ ID NO:657 and VL of SEQ ID NO:678) and a monospecific IL1RAP mAb (as described in WO2011 / 131746). Briefly, a DPBS solution (pH 7 - 7.4) of anti-IL1RAP / anti-CD3 antibody at about 1 - 20 mg / mL and 75 mM 2-mercaptoethanolamine (2-MEA) are mixed together at a molar ratio of 1.08:1 and incubated at 25°C - 37°C for 2 - 6 hours, and then 2-MEA is removed by dialysis, diafiltration, or tangential flow filtration to remove the reducing agent and enable ...

Claims

1. An isolated recombinant multispecific antibody that comprises a first antigen-binding domain that specifically binds CD3, wherein the first antigen-binding domain that specifically binds CD3 comprises: a heavy chain and a light chain, the heavy chain comprising: heavy chain complementarity-determining region (HCDR) 1 shown by the amino acid sequence of SEQ ID NO: 662, HCDR2 shown by the amino acid sequence of SEQ ID NO: 663, and HCDR3 shown by the amino acid sequence of SEQ ID NO: 664, and the light chain comprising: light chain complementarity-determining region (LCDR) 1 shown by the amino acid sequence of SEQ ID NO: 671, LCDR2 shown by the amino acid sequence of SEQ ID NO: 673, and LCDR3 shown by the amino acid sequence of SEQ ID NO:

690.

2. The isolated recombinant multispecific antibody according to claim 1, wherein the first antigen-binding domain that specifically binds CD3 comprises: a heavy chain variable region comprising SEQ ID NO: 652 and a light chain variable region comprising SEQ ID NO: 661; a heavy chain comprising SEQ ID NO: 640 and a light chain comprising SEQ ID NO: 676; a heavy chain variable region comprising SEQ ID NO: 657 and a light chain variable region comprising SEQ ID NO: 678; or a heavy chain comprising SEQ ID NO: 675 and a light chain comprising SEQ ID NO:

677.

3. The isolated recombinant multispecific antibody according to claim 1, wherein the first antigen-binding domain that specifically binds CD3 specifically binds cynomolgus monkey or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of 300 nM or less.

4. The isolated recombinant multispecific antibody according to claim 3, wherein the binding affinity is 100 nM or less.

5. The isolated recombinant multispecific antibody according to claim 3, wherein the binding affinity is measured by flow cytometry or by Proteon surface plasmon resonance using a ProteOn XPR36 system at +25°C.

6. The isolated recombinant multispecific antibody according to claim 1, wherein the antibody has one, two, three, or four of the following characteristics: a) binds human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 300 nM or less and binds HEK cells expressing cynomolgus monkey CD3 with a calculated EC50 of 300 nM or less, wherein the difference in the calculated EC50 between binding to CD3+ T lymphocytes and binding to HEK cells expressing cynomolgus monkey CD3 is less than 5-fold, and wherein the calculated EC50 is measured in a whole cell binding assay using flow cytometry at 0°C; b) binds to recombinant CD3d (SEQ ID NO: 691) from human, or binds to recombinant CD3e (SEQ ID NO: 636) from human, or binds to recombinant CD3d (SEQ ID NO: 692) from cynomolgus macaque, or binds to recombinant CD3e (SEQ ID NO: 693) from cynomolgus macaque, with an equilibrium dissociation constant (K D ) of 300 nM or less; D measured using a ProteOn XPR36 system by Proteon surface plasmon resonance at +25 °C; c) binds residues 1-6 of CD3e as determined by X-ray crystallography; or d) activates T cells or induces CD69 expression to a similar extent as cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay.

7. The isolated recombinant multispecific antibody according to claim 1, comprising at least one substitution in the antibody constant domain, said at least one substitution comprises: a) a heavy chain substitution of K409R, F405L or F405L and R409K; b) a heavy chain substitution of S228P, F234A and L235A; c) a heavy chain substitution of L234A, G237A, P238S, H268A, A330S and P331S, wherein the antibody is of IgG1 isotype; or d) a heavy chain substitution of S228P, wherein the antibody is of IgG4 isotype; wherein the residues are numbered according to the EU index.

8. The isolated recombinant multispecific antibody according to claim 1, wherein the antibody is human or humanized.

9. The isolated recombinant multispecific antibody according to claim 8, wherein the antibody is of IgG4 or IgG1 isotype.

10. The isolated recombinant multispecific antibody according to claim 1, wherein the antibody comprises a second antigen-binding domain that specifically binds a second antigen and a third antigen-binding domain that specifically binds a third antigen.

11. The isolated recombinant multispecific antibody according to claim 10, wherein the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) that are SEQ ID NO:652 and 661, respectively.

12. The isolated recombinant multispecific antibody according to claim 11, wherein at least one domain is of IgG1 isotype and comprises: (i) L234A and L235A substitutions; (ii) T366S, L368A and Y407V substitutions; and (iii) T366W substitution; wherein the residues are numbered according to the EU index.

13. The isolated recombinant multispecific antibody according to claim 12, wherein the second antigen is a cell surface antigen expressed on target cells other than immune effector cells.

14. The isolated recombinant multispecific antibody according to claim 13, wherein the cell surface antigen is a tumor-associated antigen.

15. The isolated recombinant multispecific antibody according to claim 12, wherein the second antigen is CD33, IL1RAP, PSMA or TMEFF2.

16. The isolated recombinant multispecific antibody according to claim 12, wherein the third antigen-binding domain is covalently bound to the constant region of the antibody.

17. The isolated recombinant multispecific antibody according to claim 13, wherein the third antigen-binding domain is a scFv.

18. The isolated recombinant multispecific antibody according to claim 12, wherein the third antigen is a cell surface antigen expressed on target cells other than immune effector cells.

19. The isolated recombinant multispecific antibody according to claim 18, wherein the cell surface antigen is a tumor-associated antigen.

20. A pharmaceutical composition comprising the recombinant multispecific antibody according to any one of the preceding claims and a pharmaceutically acceptable carrier.

21. A polynucleotide encoding a recombinant multispecific antibody according to any one of claims 1 to 19.

22. A vector comprising the polynucleotide according to claim 21.

23. A host cell comprising the vector according to claim 22.

24. A method for preparing a recombinant multispecific antibody according to any one of claims 1 to 19, comprising: culturing the host cell according to claim 23 under conditions that allow expression of the antibody, and recovering the recombinant multispecific antibody produced by the host cell.

25. Use of an isolated recombinant multispecific antibody according to any one of claims 1 to 19 in the preparation of a medicament for treating cancer in a subject, wherein the second antigen is a tumor-associated antigen.

26. Use according to claim 25, wherein the cancer is a solid tumor or a hematological malignancy.

27. Use according to claim 26, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell renal carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, pancreatic cancer, renal cancer, urothelial carcinoma or liver metastatic adenocarcinoma.

28. Use according to claim 26, wherein the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

29. Use according to claim 25, wherein the medicament further comprises a second therapeutic agent.

30. A recombinant multispecific antibody according to any one of claims 1 to 19 for use in therapy.

Images