Anti-CD3 antibodies and uses thereof

Abstract

The present invention relates to antibodies that specifically bind to 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 present invention relates to fragments of the antibodies, polynucleotides encoding the antibodies or fragments thereof, and methods of making and using the antibodies.

Description

[0001] sequence list

[0002] This application contains a sequence list that has been electronically filed in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on May 3, 2019, is named JBI5135USNP_Sequence_listing.txt and is 588KB in size. Technical Field

[0003] The disclosure herein relates to anti-CD3 antibodies and antigen-binding fragments thereof capable of specifically binding to human and non-human differentiation cluster 3 (CD3), and more specifically to anti-CD3 antibodies and antigen-binding fragments that cross-react with CD3 in non-human mammals (e.g., cynomolgus monkeys); PSMA antibodies and antigen-binding fragments thereof capable of specifically binding to human and non-human prostate-specific membrane antigen (PSMA); IL1RAP antibodies and antigen-binding fragments thereof capable of specifically binding to human and non-human IL1RAP; CD33 antibodies and antigen-binding fragments thereof capable of specifically binding to human and non-human CD33; and bispecific antibodies capable of specifically binding to CD3; PSMA; IL1RAP; CD33; CD3 and PSMA; CD3 and IL1RAP; or CD3 and CD33. This 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 conditions. Background Technology

[0004] Bispecific antibodies and antibody fragments have been explored as means of recruiting cytolytic T cells to kill tumor cells. However, the clinical use of many bispecific antibodies that recruit T cells is limited by challenges including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing problems. Therefore, there is a great need for bispecific antibodies that recruit cytolytic T cells to kill tumor cells exhibiting reduced toxicity and favorable manufacturing characteristics.

[0005] The human CD3 T-cell antigen receptor protein complex consists of six distinct chains: one CD3γ chain (SwissProtP09693), one CD3δ chain (SwissProtP04234), two CD3ε chains (SwissProtP07766), and a CD3ζ chain homodimer (SwissProtP20963) (εγ:εδ:ζζ). This homodimer associates with the α and β chains of the T-cell receptor. This complex plays a crucial 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 essential for the immune response. Summary of the Invention

[0006] This article provides an isolated recombinant anti-CD3 antibody or its antigen-binding fragment thereof, comprising: a heavy chain and a light chain, wherein the heavy chain comprises: a heavy chain complementarity-determining region (HCDR)1 containing SEQ ID NO:662, an HCDR2 containing SEQ ID NO:663, and an HCDR3 containing SEQ ID NO:664; the light chain comprises: a light chain complementarity-determining region (LCDR)1 containing SEQ ID NO:671, an LCDR2 containing SEQ ID NO:673, and an LCDR3 containing SEQ ID NO:690; a heavy chain variable region containing SEQ ID NO:652 and a light chain variable region containing SEQ ID NO:661; a heavy chain containing SEQ ID NO:640 and a light chain containing SEQ ID NO:676; or comprising: a heavy chain and a light chain, wherein the heavy chain comprises: an HCDR1 containing SEQ ID NO:662, an HCDR2 containing SEQ ID NO:663, and an HCDR3 containing SEQ ID NO:664; and the light chain comprises: a heavy chain containing SEQ ID NO:662, an HCDR2 containing SEQ ID NO:663, and an HCDR3 containing SEQ ID NO:664; and the light chain comprises: a heavy chain containing SEQ ID NO:662, an HCDR1 ... LCDR1 containing SEQ ID NO:773, LCDR2 containing SEQ ID NO:673, and LCDR3 containing SEQ ID NO:690; a heavy chain variable region containing SEQ ID NO:657 and a light chain variable region containing SEQ ID NO:678; or a heavy chain containing SEQ ID NO:675 and a light chain containing SEQ ID NO:678.

[0007] It also provides isolated recombinant anti-CD3 antibodies or antigen-binding fragments thereof that specifically bind to cynomolgus monkey (Macacafascicularis) or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of about 300 nM or less.

[0008] In some embodiments, the isolated recombinant anti-CD3 antibody or its antigen-binding fragment has one, two, three, or four of the following characteristics:

[0009] • Human and cynomolgus monkey CD3+ T lymphocytes were bound with a calculated EC50 of 20 nM or less, and cynomolgus monkey CD3-expressing HEK cells were bound with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between the bound CD3+ T lymphocytes and the bound cynomolgus monkey CD3-expressing HEK cells was less than 5-fold, and wherein the calculated EC50 was measured in a whole-cell binding assay by flow cytometry at 0°C.

[0010] • Recombined CD3d from humans (SEQ ID NO:691), or recombined CD3e from humans (SEQ ID NO:636), or recombined CD3d from cynomolgus monkeys (SEQ ID NO:692) with an equilibrium dissociation constant (KD) of 12 nM or less, wherein KD was determined using the Proteon surface plasmon resonance determination ProteOnXPR36 system at +25 °C;

[0011] • Residues 1-6 of CD3e, as determined by X-ray crystallography; or

[0012] • Activate T cells or induce CD69 expression to a level similar to cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0013] In some embodiments, the antibody or its antigen-binding fragment described herein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID Nos: 662, 663, 664, 671, 673, and 690, respectively; VH and VL of SEQ ID Nos: 652 and 661, respectively; or HC and LC of SEQ ID Nos: 640 and 676, respectively.

[0014] In some embodiments, the antibodies or antigen-binding fragments described herein comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID Nos: 662, 663, 664, 773, 673, and 690, respectively; VH and VL of SEQ ID Nos: 657 and 678, respectively; or HC and LC of SEQ ID Nos: 675 and 677, respectively.

[0015] This article further describes a bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen, wherein the first domain comprises the antibody or its antigen-binding fragment described herein.

[0016] The bispecific antibody described herein may comprise a first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as specified in SEQ ID NO: 662, 663, 664, 671, 673, and 670, respectively; and a second domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as specified in SEQ ID NO: 697, 683, 698, 699, 792, and 686, respectively.

[0017] The bispecific antibody described herein may comprise a first domain comprising VH and VL of SEQ ID NO:652 and 661, respectively; and a second domain comprising VH and VL of SEQ ID NO:681 and 682, respectively.

[0018] The bispecific antibody described herein may comprise a first domain comprising HC and LC of SEQ ID NO: 640 and 676, respectively; and a second domain comprising HC and LC of SEQ ID NO: 679 and 680, respectively.

[0019] The bispecific antibody described herein may comprise a first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as specified in SEQ ID NO: 662, 663, 664, 671, 673, and 670, respectively; and a second domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as specified in SEQ ID NO: 704, 705, 706, 707, 708, and 709, respectively.

[0020] The bispecific antibody described herein may comprise a first domain comprising VH and VL of SEQ ID NO:652 and 661, respectively; and a second domain comprising VH and VL of SEQ ID NO:702 and 703, respectively.

[0021] The bispecific antibody described herein may comprise a first domain comprising HC and LC of SEQ ID NO: 640 and 676, respectively; and a second domain comprising HC and LC of SEQ ID NO: 700 and 701, respectively.

[0022] This disclosure also provides an isolated bispecific CD3×PSMA antibody comprising a first domain that binds to cells expressing recombinant cynomolgus monkey 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.

[0023] In some implementations, the bispecific CD3×PSMA antibody includes a first domain, which:

[0024] • Human and cynomolgus monkey CD3+ T lymphocytes were bound with a calculated EC50 of 20 nM or less, and cynomolgus monkey CD3-expressing HEK cells were bound with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between the bound CD3+ T lymphocytes and the bound cynomolgus monkey CD3-expressing HEK cells was less than 5-fold, and wherein the calculated EC50 was measured in a whole-cell binding assay by flow cytometry at 0°C.

[0025] • Recombined CD3d from humans (SEQ ID NO:691), or recombined CD3e from humans (SEQ ID NO:636), or recombined CD3d from cynomolgus monkeys (SEQ ID NO:692), or recombined CD3e from cynomolgus monkeys (SEQ ID NO:693) with an equilibrium dissociation constant (KD) of 12 nM or less, wherein KD was measured using the Proteon surface plasmon resonance determination ProteOnXPR36 system at +25 °C;

[0026] • If detected by peptide profiling analysis, it shows no oxidation of methionine or tryptophan, or no deamidation of asparagine, or no isomerization of asparagine;

[0027] • Residues 1-6 of CD3e, as determined by X-ray crystallography; or

[0028] • Activate T cells or induce CD69 expression to a level similar to cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0029] The present invention also describes pharmaceutical compositions comprising the antibodies described herein and pharmaceutically acceptable carriers.

[0030] This disclosure also provides methods for generating the antibodies described herein, including culturing a host cell containing a vector under antibody-expressing conditions, and recovering the antibody generated by the host cell, the vector containing a polynucleotide encoding the antibody described herein or an antigen-binding fragment thereof. A method for generating bispecific CD3×PSMA antibodies may include mixing a monospecific bivalent CD3 antibody having two identical HC1 and two identical LC1 with a monospecific bivalent PSMA antibody having two identical HC2 and two identical LC2 in 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 containing HC1, LC1, HC2, and LC2.

[0031] The present invention also describes a method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of the isolated antibody to the subject in need for a time sufficient to treat the cancer.

[0032] This disclosure also provides kits containing the antibodies described herein. The kits may also include reagents for detecting the antibodies and instructions for use. Attached Figure Description

[0033] The invention and the detailed embodiments described below will be further understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the antibodies and methods of this disclosure, the drawings show exemplary embodiments of the antibodies and methods; however, the antibodies and methods are not limited to the specific embodiments disclosed. In the drawings:

[0034] Figure 1A and Figure 1B This shows the anti-CD3 antibody produced in OmniRat. The active anti-CD3 mAb produced in OmniRat is then compared with VH( Figure 1A ) and VL ( Figure 1B The sequences were compared with pedigree sequences from IMGT. 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 appearance order.

[0035] Figure 2 A cell-based binding assay was demonstrated to evaluate the binding ability of a single rat hybridoma supernatant to purified human CD3+ T lymphocytes.

[0036] Figure 3 A cell-based binding assay was shown to evaluate the binding ability of a single rat hybridoma supernatant to purified CD3+ T lymphocytes from cynomolgus monkeys.

[0037] Figure 4 The results of a competitive assay of hybridoma supernatant are shown, which evaluated 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 CD3.

[0038] Figure 5 The representative binding curves of anti-CD3 antibodies on primary human T cells are shown.

[0039] Figure 6The representative competitive binding curves of anti-CD3 antibody conjugated with SP34-2 conjugated with AlexaFluor488 on primary human T cells are shown.

[0040] Figure 7 The binding of light chain (LC) engineered BLW-2E6mAb to primary human T cells was demonstrated.

[0041] Figure 8 The binding of heavy chain (HC) engineered BLW-2E6 to primary human T cells was demonstrated.

[0042] Figure 9 This shows Alexa Fluor in human T cells as determined by FACS analysis. TM (AF)488CD3 saturation. The average fluorescence intensity values ​​were plotted as a function of antibody molecule concentration. Kd values ​​for each donor were derived and averaged. The saturation binding constant (KdT) for human T cells was derived as 5.6 ± 1.0 nM (n = 4) and used in this paper to determine Kd binding affinity. One donor was excluded because they did not meet the minimum 60% survival criterion during the analysis. The “LS” identifier in the legend refers to an individual human T cell donor.

[0043] Figure 10 The inhibition curves of the bivalent anti-CD3 antibodies CD3B376 and CD3B450 competitively binding to the Alexa Fluor 488SP-34 anti-CD3 antibody are shown. The IC50 values ​​were derived as 29 nM and 60 nM, respectively.

[0044] Figure 11 Sensing plot of the BLW-2E6 variant combined with hCD3ε(1-27)-Tn25 is shown.

[0045] Figures 12A to 12E The anti-CD3 antibody CD3B376 (as determined by DSC) is shown. Figure 12A CD3B450 Figure 12B ), CD3B389 ( Figure 12C ), CD3B467 ( Figure 12D The thermal stability of ) and the superposition of the thermograms of all candidates ( Figure 12E ).

[0046] Figures 13A to 13B CD3B376 and CD3B389 are shown. Figure 13A Comparison of the thermal stability of superimposed thermograms of anti-CD3 antibodies; superimposed thermograms of CD3B450 and CD3B467 ( Figure 13B ).

[0047] Figure 14The binding of a subset of affinity-matured PSMA×CD3 bispecific antibodies to LNCAP cells was demonstrated.

[0048] Figure 15 The binding of a subset of affinity-matured PSMA×CD3 bispecific antibodies to LNCAP cells was demonstrated.

[0049] Figure 16 The binding results of the affinity-matured PSMA×CD3 bispecific antibody to PSMA-negative PC3 cells are shown.

[0050] Figure 17 The results of bispecific Abs maturing with PSMA×CD3 affinity are shown in the functional cell killing assay.

[0051] Figure 18 The antibody-antigen interaction in the CD3B334:CD3 complex is shown. CD3 residues are in ellipses, and CD3B334 residues are in boxes.

[0052] Figure 19 The assay, based on primary human and cynomolgus monkey T cells, is shown. This assay was used to determine the ability of hybridomas to hit activated T cells, as measured by CD69 activation.

[0053] Figure 20 This study demonstrates the antitumor efficacy of PS3B79 in LnCAPAR.TB human prostate xenografts from T-cell humanized NSG mice. Subcutaneous LnCAPAR.TB tumors were measured twice weekly, and results are expressed as mean tumor volume in mm. 3 ±SEM represents (*, p<0.0001).

[0054] Figure 21 This study demonstrates the antitumor efficacy of PS3B90 in LnCAPAR.TB human prostate xenografts from T-cell humanized NSG mice. Subcutaneous LnCAPAR.TB tumors were measured twice weekly, and results are expressed as mean tumor volume in mm. 3 ±SEM indicates (*, p<0.001).

[0055] Figures 22A to 22D The titration curves for anti-PSMA phage panning hits with human LNCaP cells are shown. Figure 22A Titration curves for hits G9-PSMM18, M25, M50, M52, M56, M57 and M59 are shown; Figure 22B The titration curves for M52 and M110 are shown; Figure 22C The titration curves for M85, M87, and M81 are shown; and Figure 22D The titration curves for M52 and M84 are shown. Figure 22D In this study, Fab expression in the supernatant of mammalian expression was normalized using octet, and titration was performed on human LNCAP, PSMG5 (cynomolgus monkey PSMAHEK), or PSMG9 (chimpanzee PSMAHEK) cells using flow cytometry. The relationship between geometric mean fluorescence intensity (GeoMFI) and Fab concentration was plotted using GraphPadPrizm.

[0056] Figures 23A to 23D Titration curves of anti-PSMA phage panning hits against HEK cells expressing chimpanzee PSMA are shown. (Titration curves of mammalian Fab supernatant for anti-PSMA phage panning hits against chimpanzee PSMA HEK). Figure 23A Titration curves for hits G9-PSMM18, M25, M50, M52, M56, M57 and M59 are shown; Figure 23B The titration curves for M52 and M110 are shown; Figure 23C The titration curves for M81, M52, M85, and M87 are shown; and Figure 23D The titration curves for M52 and M84 are shown. Figure 23D In this study, Fab expression in the supernatant of mammalian expression was normalized using octet, and titration was performed on human LNCAP, PSMG5 (cynomolgus monkey PSMAHEK), or PSMG9 (chimpanzee PSMAHEK) cells using flow cytometry. The relationship between geometric mean fluorescence intensity (GeoMFI) and Fab concentration was plotted using GraphPadPrizm.

[0057] Figures 24A to 24D Titration curves of anti-PSMA phage panning hits against HEK cells expressing cynomolgus monkey PSMA are shown. (Titration curves of mammalian Fab supernatant for anti-PSMA phage panning hits against cynomolgus monkey PSMA HEK). Figure 24A Titration curves for hits G9-PSMM18, M25, M50, M52, M56, M57 and M59 are shown; Figure 24B The titration curves for M52 and M110 are shown; Figure 24C The titration curves for M81, M52, M85, and M87 are shown; and Figure 24D The titration curves for M52 and M84 are shown. Figure 24D In this study, Fab expression in the supernatant of mammalian expression was normalized using octet, and titration was performed on human LNCAP, PSMG5 (cynomolgus monkey PSMAHEK), or PSMG9 (chimpanzee PSMAHEK) cells using flow cytometry. The relationship between geometric mean fluorescence intensity (GeoMFI) and Fab concentration was plotted using GraphPadPrizm.

[0058] Figure 25 The overall structure of PSMM84Fab bound to the human PSMAECD homodimer is shown.

[0059] Figure 26 A close-up view of the main interactions between PSMA and the PSMB83 light chain is shown.

[0060] Figure 27 A close-up view of the main interactions between PSMA and the PSMB83 heavy chain is shown.

[0061] Figure 28 A comparison of epitope residues of PSMB83 within the sequence of PSMA in humans (SEQ ID NO:719), mice (SEQ ID NO:720), and cynomolgus monkeys (SEQ ID NO:721) is shown. Epitope residues are shown in shaded areas, and sequence differences are indicated by underlining.

[0062] Figure 29 The complementary residues of PSMB83 are shown. CDRs are shown underlined, and complementary residues are shown in shaded areas. Figure 29 SEQ ID NO:722-727 are disclosed in appearance order.

[0063] Figure 30 An interaction diagram showing direct contact between PSMA and PSMM84 is presented. Van der Waals interactions are shown as dashed lines, and H bonds are indicated by solid arrows pointing towards the main chain atoms.

[0064] Figure 31 The expression levels of the anti-PSMAFab clone derived from PSMM84 are shown compared to those of the parent PSMB83. The original luminescence number is plotted against the logarithmic concentration.

[0065] Figure 32 The binding of the anti-PSMAFab clone derived from PSMB83 to human PSMA is shown compared to the binding of the parental PSMM84. The original luminescence number is plotted against the logarithmic concentration.

[0066] Figure 33 The binding of the PSMB83-derived anti-PSMAFab clone to cynomolgus monkey PSMA is shown compared to the binding to the parent PSMB83. The original luminescence number is plotted against the logarithmic concentration.

[0067] Figure 34 The in vitro T-cell cytotoxicity mediated by IC3B19 and IC3B34 in LAMA-84 cells in whole blood after 48 hours is shown. The concentrations of IC3B19 and IC3B34 are provided in the table at the bottom of the figure.

[0068] Figure 35 The figure shows the in vitro T cell activation mediated by 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.

[0069] Figures 36 through 55 illustrate the binding of IC3B19 and IC3B34-mediated T cell and IL1RAP+ target cell line LAMA-84 (endogenous and exogenously added tumor cells). Cytotoxicity of the supernatant with 10 pro-inflammatory cytokines from whole blood (n = 15 donors) and T cell activation assays of the exogenously added LAMA-84 IL1RAP+ tumor cell line were assessed. Statistically significant differences in these figures are indicated in bold text.

[0070] Figures 36A to 36B This demonstrates the release of T cell IL-1β mediated by IC3B19 and IC3B34 24 hours later. Figure 36A ) and the corresponding 4PL regression parameter estimates ( Figure 36B ).

[0071] Figures 37A to 37B This demonstrates the release of T cell IL-1β mediated by IC3B19 and IC3B34 48 hours later. Figure 37A ) and the corresponding 4PL regression parameter estimates ( Figure 37B ).

[0072] Figures 38A to 38B This demonstrates the release of T cell IL-2 mediated by IC3B19 and IC3B34 24 hours later. Figure 38A ) and the corresponding 4PL regression parameter estimates ( Figure 38B ).

[0073] Figures 39A to 39B This demonstrates the release of T cell IL-2 mediated by IC3B19 and IC3B34 48 hours later. Figure 39A ) and the corresponding 4PL regression parameter estimates ( Figure 39B ).

[0074] Figures 40A to 40B This demonstrates the release of T cell IL-4 mediated by IC3B19 and IC3B34 24 hours later. Figure 40A ) and the corresponding 4PL regression parameter estimates ( Figure 40B ).

[0075] Figure 41 The study showed that T cell IL-4 release was mediated by IC3B19 and IC3B34 48 hours later.

[0076] Figures 42A to 42BThis demonstrates the release of T cell IL-6 mediated by IC3B19 and IC3B34 24 hours later. Figure 42A ) and the corresponding 4PL regression parameter estimates ( Figure 42B ).

[0077] Figure 43 The study showed that T cell IL-6 release was mediated by IC3B19 and IC3B34 48 hours later.

[0078] Figure 44 The study showed that T cell IL-8 release was mediated by IC3B19 and IC3B34 24 hours later.

[0079] Figure 45 The study showed that T cell IL-8 release was mediated by IC3B19 and IC3B34 48 hours later.

[0080] Figures 46A to 46B This demonstrates the release of T cell IL-10 mediated by IC3B19 and IC3B34 24 hours later. Figure 46A ) and the corresponding 4PL regression parameter estimates ( Figure 46B ).

[0081] Figures 47A to 47B This demonstrates the release of T cell IL-10 mediated by IC3B19 and IC3B34 48 hours later. Figure 47A ) and the corresponding 4PL regression parameter estimates ( Figure 47B ).

[0082] Figure 48 The study showed that IL-12p70 release from T cells was mediated by IC3B19 and IC3B34 24 hours later.

[0083] Figure 49 The study showed that IL-12p70 release from T cells was mediated by IC3B19 and IC3B34 48 hours later.

[0084] Figure 50 The study showed that T cell IL-13 release was mediated by IC3B19 and IC3B34 24 hours later.

[0085] Figures 51A to 51B This demonstrates the release of T cell IL-13 mediated by IC3B19 and IC3B34 48 hours later. Figure 51A ) and the corresponding 4PL regression parameter estimates ( Figure 51B ).

[0086] Figures 52A to 52B This demonstrates the release of IFN-γ from T cells mediated by IC3B19 and IC3B34 24 hours later. Figure 52A ) and the corresponding 4PL regression parameter estimates ( Figure 52B).

[0087] Figures 53A to 53B This demonstrates the release of IFN-γ from T cells mediated by IC3B19 and IC3B34 48 hours later. Figure 53A ) and the corresponding 4PL regression parameter estimates ( Figure 53B ).

[0088] Figures 54A to 54B This demonstrates the release of TNF-α from T cells mediated by IC3B19 and IC3B34 24 hours later. Figure 54A ) and the corresponding 4PL regression parameter estimates ( Figure 54B ).

[0089] Figures 55A to 55B This demonstrates the release of TNF-α from T cells mediated by IC3B19 and IC3B34 48 hours later. Figure 55A ) and the corresponding 4PL regression parameter estimates ( Figure 55B ).

[0090] Figure 56 The assay showed that bispecific antibodies (IAPB57×B23B49 or B23B39×CD3B219) induced target-specific cytotoxicity in NCI-H1975 cells using IC3B19 and IC3B34, instead of the blank arm. In this assay, the cytotoxic EC50 varied three-fold between IC3B19 and IC3B34, with values ​​of 0.018 nM and 0.057 nM, respectively.

[0091] Figure 57 The antitumor efficacy of IAPB57×CD3B376 in H1975 human NSCLC xenografts from T-cell humanized NSG mice is demonstrated. Subcutaneous H1975 tumors were measured twice weekly, and results are expressed as mean tumor volume in mm3 ± SEM, *p<0.0001.

[0092] Figure 58 A comparison of the isoelectric points of various anti-PSMA constructs is shown.

[0093] Figure 59 The wavelength variation compared to the CNTO5825 control molecule is plotted. The control CNTO607 exhibits characteristic strong self-interactions. Error bars represent the standard deviation of three repeated measurements.

[0094] Figure 60 The comparison of retention times on the IgG column and the control column in the anti-PSMA cross-interaction assay is shown.

[0095] Figures 61A to 61BThe in vitro assessment of cytotoxicity and T-cell activation of blast cells in whole blood from fresh AML patients using CD33×CD3 bispecific antibodies against CD3B219 and CD3B376 (anti-CD3 arms) is shown. Figure 61A The percentage of total cytotoxicity in AML cells using either the CD33 bispecific antibody or the CD3× blank control is shown. Figure 61B T cell activation induced by CD33 bispecific antibody or CD3× blank control is shown. No Fc blocker was added.

[0096] Figures 62A to 62C This demonstrates a CD33×CD3 T cell-mediated cytotoxicity assay. The CD33×CD3 bispecific antibody against CD3 arm CD3B219 and anti-CD3B376 was used with human panT cells and against the CD33 SNP rs12459419 mutation (wild-type KG1). Figure 62A ), hybrid type (SH2, Figure 62B ) or homozygous (OCIAML3, Figure 62C The AML cell lines were incubated together. After incubation at 37°C and 5% CO2 for 48 hours, the cytotoxicity of total tumor cells was measured by flow cytometry.

[0097] Figures 63A to 63B The ex vivo assessment of the cytotoxicity of exogenously added MOLM-13 cells to normal healthy human whole blood (N=6 donors) with C33B904 antibody paired with CD3B219 or CD3B376 is shown: the percentage of MOLM-13 cells with cytotoxicity after 48 hours using CD33×CD3 bispecific antibody and corresponding blank×CD3 control. Figure 63A ) and CD33 + CD14 + cytotoxicity percentage of monocytes ( Figure 63B ).

[0098] Figures 64A to 64B The in vitro evaluation of cytotoxicity and T-cell activation of monocytes from fresh whole blood from six normal cynomolgus monkey donors using CD33×CD3 bispecific antibodies against CD3 arms CD3B219 and CD3B376 is shown. Figure 64A The following diagram shows CD33 using a CD33 bispecific antibody or its CD3× blank control. + CD14 + The percentage of total cytotoxicity in cynomolgus monkey mononuclear cells. Figure 64B The study demonstrates T cell activation induced by the CD33 bispecific antibody or its CD3× blank control. No Fc blocker was added.

[0099] Figure 65This study demonstrates the antitumor efficacy of C3CB189 in MOLM-13 human AML xenografts from T-cell humanized NSG mice. Diffuse MOLM-13 tumors were bioluminescently imaged twice weekly, and results are presented as mean luminescence (p / s / cm²). 2 / sr)±SEM (n=8-10 / group) is expressed as (n=8-10 / group). *p≤0.0001 for treatment and control, calculated by two-way ANOVA and Bonferroni test.

[0100] Figure 66 The survival rates of animals treated with C3CB189 in MOLM-13 human AML xenografts in T-cell humanized NSG mice are shown. Survival rates of MOLM-13-carrying mice are graphically represented using Kaplan-Meier curves and evaluated by the Mantel-Cox test. *p≤0.0001 for treatment groups vs. control groups.

[0101] Figure 67 This study demonstrates the antitumor efficacy of C3CB88 in MOLM-13 human AML xenografts from T-cell humanized NSG mice. Diffuse MOLM-13 tumors were bioluminescently (BLI) images performed twice weekly, and results are presented as mean luminescence (p / s / cm²). 2 / sr)±SEM (n=8-10 / group) is expressed as (n=8-10 / group). *p≤0.0001 for treatment and control, calculated by two-way ANOVA and Bonferroni test.

[0102] Figure 68 The survival rates of animals treated with C3CB88 in MOLM-13 human AML xenografts in T-cell humanized NSG mice are shown. Survival rates of MOLM-13-carrying mice are graphically represented using Kaplan-Meier curves and evaluated by the Mantel-Cox test. *p≤0.05 for treatment groups vs. control groups.

[0103] Figure 69 The alignment of selected anti-TMEFF2 antibody heavy chain variable regions (VH) is shown. VH regions are identified at the beginning of each row by their SEQ ID NO:.

[0104] Figure 70 Alignment of selected anti-TMEFF2 antibody light chain variable regions (VL) is shown. VH regions are identified at the beginning of each row by their SEQ ID NO:.

[0105] Figure 71This study demonstrates the reduction in mean tumor volume per mouse treated with 0.5 mg / kg TMCB132 in an in vitro LnCaP prostate cancer model in male NGS mice.

[0106] Figure 72 This study demonstrates the efficacy of TMEB762×CD3B376 in established LNCaP xenografts in humanized T-cell NSG mice.

[0107] Figure 73 The study demonstrated T cell activation in LnCaP prostate cancer cells in response to TMCB132 administration.

[0108] Figure 74 The T cell-mediated cytotoxicity of TMCB132 was demonstrated.

[0109] Figure 75 The antitumor efficacy of TMCB132 in humanized T-cell mice was demonstrated. Detailed Implementation

[0110] All publications cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference as if they were given in their entirety.

[0111] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is 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 pertains.

[0112] While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, exemplary materials and methods are described herein. The following terminology will be used in describing and claiming the invention.

[0113] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0114] "Specific binding," "specifically binding," or simply "binding" refers to an antibody binding to a specific antigen or an epitope within that antigen with a higher affinity than it has for other antigens. Typically, antibodies bind at an affinity of approximately 5 × 10⁻⁶. -8 M or smaller (e.g., about 1×10⁻⁶) -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or approximately 1×10 -12 The equilibrium dissociation constant (K) of M or lessD ) binds to the antigen or an epitope within the antigen, usually this K D This antibody binds to the K+ of non-specific antigens (such as BSA, casein). D At most one percent. The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to the same antigen from other species (homologous) (such as humans or monkeys, e.g., cynomolgus (cyno) and pantroglodytes (chimpanzee (chimp)). While monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.

[0115] "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, dimer, tetramer, or polymeric antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of immunoglobulin molecules containing an antigen-binding site with desired specificity. A "full-length antibody molecule" comprises two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, as well as their polymers (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (composed of 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 and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0116] The complementarity-determining region (CDR) is the region of the antibody that binds to the antigen. CDRs can be defined using various descriptions, such as those by 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 B Mol Biol, Vol. 263, pp. 800-815). The correspondence between various depictions and variable region numbering is described (see, for example, Lefranc et al., 2003, DevCompImmunol, Vol. 27, pp. 55-77; Honegger and Pluckthun, 2001, JMolBiol, Vol. 309, pp. 657-670; International Immunogenetics (IMGT) Database; Web resource, http: / / www_imgt_org). Available programs (such as abYsis of UCL BusinessPLC) can be used to depict CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” include CDRs as defined by any of the aforementioned methods (Kabat, Chothia, IMGT, or AbM).

[0117] Immunoglobulins can be designated into five major types based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of their constant domain, the antibody light chain of any vertebrate species can be designated into one of two completely different types: κ and λ.

[0118] "Antigen-binding fragment" refers to the portion of an immunoglobulin molecule that binds to an antigen. Antigen-binding fragments can be synthetic, enzymatically obtained, or genetically engineered polypeptides, and include VH, VL, VH and VL, Fab, F(ab')2, Fd, and Fv fragments, domain antibodies (dAbs) consisting of a VH domain or a VL domain, shark variable IgNAR domains, camelified VH domains, and minimal recognition units consisting of amino acid residues of the CDR of a mimic antibody (such as the FR3-CDR3-FR4 moiety, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3). The VH and VL domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, wherein, where the VH and VL domains are expressed by separate single-chain antibody constructs, the VH / VL domains can be paired intramolecularly or intermolecularly to form monovalent antigen-binding sites, such as single-chain Fv (scFv) or bivalent antibodies; as described, for example, in international patent publications WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047.

[0119] "Monoclonal antibody" refers to antibodies obtained from a substantially homogeneous population of antibody molecules; that is, the individual antibodies constituting the population are identical except for possibly well-known alterations (such as removal of a 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 antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic 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.

[0120] "Isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to an antigen that is substantially free of antibodies that specifically bind to antigens other than that antigen). In the case of a bispecific CD3 antibody, the bispecific antibody specifically binds to both CD3 and the second antigen. "Isolated antibody" encompasses antibodies isolated to higher purities, such as antibodies with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0121] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one frame is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the frame such that the frame may not be an exact copy of the expressed human immunoglobulin or the germline gene sequence of a human immunoglobulin.

[0122] "Human antibody" refers to an antibody optimized to produce 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, 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 immunoglobulins or rearranged immunoglobulin genes, the human antibody contains both heavy-chain and light-chain variable regions "derived" from human-origin sequences. Exemplary systems of this kind are libraries of human immunoglobulin genes displayed on bacteriophages, and transgenic nonhuman 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 frame or CDR, or both, "human antibody" typically contains amino acid differences compared to immunoglobulins expressed in humans. Typically, 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 with the amino acid sequence encoded by a human germline immunoglobulin gene or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain a common frame sequence obtained from human frame sequence analysis (e.g., described in Knappik et al., 2000, J Mol Biol, Vol. 296, pp. 57-86); or be bound to a synthetic HCDR3 in a human immunoglobulin gene library displayed on a bacteriophage (e.g., described in Shi et al., 2010, J Mol Biol, Vol. 397, pp. 385-396 and International Patent Publication WO2009 / 085462).

[0123] The "percentage of 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 those in the reference polypeptide sequence, after sequence alignment and the introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignments performed for the purpose of determining the percentage of amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the amino acid sequence identity % value is 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 is filed with the U.S. Copyright Office (Washington D.C., 20559) 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 source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity) is calculated as follows:

[0124] 100 multiplied by the fraction X / Y,

[0125] Where X is the number of amino acid residues that the sequence alignment program ALIGN-2 scores as identical matches in the alignment of A and B, and 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 amino acid sequence identity % from A to B is not equal to the amino acid sequence identity % from B to A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraphs.

[0126] The definition of "human antibody" does not include antibodies whose antigen-binding sites are derived from non-human species.

[0127] "Recombinant" refers to DNA, antibodies, and other proteins that are prepared, expressed, formed, or isolated through recombination when fragments from different sources are joined to produce recombinant DNA, antibodies, or proteins.

[0128] An epitope is a portion of an antigen that specifically binds to an antibody. Epitopes typically consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups, such as amino acids or polysaccharide side chains, and may possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be composed of continuous and / or discontinuous amino acids forming conformational spatial units. For discontinuous epitopes, amino acids from different portions of the linear sequence of the antigen are close together in three-dimensional space due to the folding of the protein molecule. Antibody epitopes depend on the method used to identify them.

[0129] "Complementary site" refers to the portion of an antibody that specifically binds to the antigen. Complementary sites can be linear or discontinuous, formed by the spatial relationships between non-adjacent amino acids in the antibody, rather than a linear sequence of amino acids. "Light chain complementary site" and "heavy chain complementary site," or "light chain complementary amino acid residue" and "heavy chain complementary amino acid residue," refer to the light and heavy chain residues of the antibody that contact the antigen, respectively. In general, "antibody complementary site residue" refers to those antibody amino acids that contact the antigen.

[0130] "Bispecific" refers to antibodies that specifically bind to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may have cross-reactivity with other related antigens, for example, cross-reactivity with the same antigen from other species (homologous) (such as humans or monkeys, such as cynomolgus macaques (cyno) or chimpanzees), or they may bind to epitopes shared between two or more different antigens.

[0131] "Multispecific" refers to antibodies that specifically bind to two or more different antigens or two or more different epitopes within the same antigen. Multispecific antibodies may have cross-reactivity with other related antigens, for example, cross-reactivity with the same antigen from other species (homologous) (such as humans or monkeys, such as cynomolgus macaques (cyno) or chimpanzees), or they may bind to epitopes shared between two or more different antigens.

[0132] A “variant” is a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide due to one or more modifications (e.g., one or more substitutions, insertions, or deletions).

[0133] A "vector" is a polynucleotide capable of replicating within or moving between biological systems. Vector polynucleotides typically contain elements such as origins of replication, promoters, polyadenylation signals, or selection markers, whose function is to facilitate or maintain the replication of these polynucleotides in biological systems, such as cells, viruses, animals, plants, and recombinant biological systems utilizing biological components capable of replicating vectors. Vector polynucleotides can be single-stranded or double-stranded DNA or RNA molecules, or hybrid molecules of these.

[0134] "Expression vector" refers to a vector that can be used in biological or regenerated biological systems to guide the translation of polypeptides encoded by polynucleotide sequences present in the expression vector.

[0135] A "polynucleotide" is a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical linkages. Double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA, are typical examples of polynucleotides. A "polynucleotide" can also refer to a synthetic molecule comprising a chain of nucleotides covalently linked by a phosphate-sugar backbone or other equivalent covalent chemical linkages. cDNA is an exemplary synthetic polynucleotide.

[0136] A polypeptide or protein is a molecule that contains at least two amino acid residues linked together by peptide bonds to form a polypeptide. Small polypeptides with fewer than 50 amino acids can be called peptides.

[0137] 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 a laser to emit light of different wavelengths (Adan et al., Critical Reviews in Biotechnology, 2016, pp. 1549-7801).

[0138] "Anti-idiotypic (anti-Id) antibody" is an antibody that recognizes an antigenic determinant (e.g., complementary site or CDR) of an antibody. Methods for producing or preparing anti-idiotypic antibodies are well known in the art (Lathey, J. et al., Immunology, 1986, Vol. 57, No. 1, pp. 29-35). Anti-Id antibodies can be antigen-blocking or non-blocking. Antigen-blocking anti-Id antibodies can be used to detect free antibodies (e.g., CD3) in a sample. Non-blocking anti-Id antibodies can be used to detect total antibodies (free, partially bound to, or fully bound to the antigen) in a sample. Anti-Id antibodies can be prepared by immunizing an animal in which the anti-Id antibody is being prepared with the antibody. In some embodiments described herein, anti-idiotypic antibodies are used to detect the level of therapeutic antibodies in a sample.

[0139] Anti-Id antibodies can also be used as immunogens to induce an immune response in another animal, thereby producing so-called anti-anti-Id antibodies. Anti-anti-Id antibodies may be epitope identical to the original mAb that induced the anti-Id antibody. Therefore, other clones expressing the same specific antibody can be identified by using antibodies targeting the idiotype determinants of the monoclonal antibody. Anti-Id antibodies can be modified (thus producing anti-Id antibody variants) and / or derived by any suitable technique, such as those described elsewhere herein with respect to anti-CD3 antibodies.

[0140] 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.

[0141] Throughout this specification, "CD3-specific," "specifically binding to CD3," or "anti-CD3 antibody" refers to antibodies that specifically bind to the CD3-ε polypeptide (SEQ ID NO: 635), including antibodies that specifically bind to the CD3-ε extracellular domain (ECD) (SEQ ID NO: 636). CD3-ε, together with CD3-γ, -δ, and -ζ, and the T cell receptor α / β and γ / δ heterodimers, forms the T cell receptor-CD3 complex. This complex plays a crucial 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 immune responses.

[0142] SEQ ID NO:635 (human CD3ε)

[0143] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGS KPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0144] SEQ ID NO:636 (human CD3ε extracellular domain)

[0145] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0146] As used herein, the terms “interleukin-1 receptor helper protein,” “IL1RAP,” and “IL1-RAP” specifically include human IL1RAP proteins, such as 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 is also referred to in the scientific literature as IL1R3, C3orf13, FLJ37788, IL-1RAcP, and EG3556.

[0147] "CD33" refers to the differentiation cluster 33 protein. CD33 is a 67 kilodalton (kD) single-pass transmembrane glycoprotein and a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family. CD33 is primarily considered a myeloid-like differentiation antigen, expressed at low levels in myeloid-like progenitor cells, neutrophils, and macrophages, but highly expressed in circulating monocytes and dendritic cells. The human CD33 extracellular domain (UniprotP20138) (SEQ ID NO:636) and cynomolgus monkey CD33 (XP_005590138.1) are examples of proteins used in generating CD33-specific antibodies according to this disclosure.

[0148] "TMEFF2" refers to a human transmembrane protein containing EGF-like and two follistatin-like domains, also known as tomoregulin2. The amino acid sequence of the 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 the full-length TMEFF2. The extracellular domain of TMEFF2 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 "proximal membrane region" of TMEFF2 refers to the TMEFF2 region of SEQ ID NO:629, which encompasses the EGF domain and the NC-terminal linker region (e.g., residues 230-320 of the full-length human TMEFF2 in SEQ ID NO:77). Unless explicitly stated to be derived from a non-human species, all references to proteins, peptides, and protein fragments herein are intended to refer to the human form of the corresponding protein, peptide, or protein fragment. Therefore, unless a non-human species is specified, such as “mouse TMEFF2” or “monkey TMEFF2”, “TMEFF2” means human TMEFF2.

[0149] SEQ ID NO:77 (Full-length human TMEFF2)

[0150] MVLWESPRQCSSWTLCEGFCWLLLLLPVMLLIVARPVKLAAFPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCS QTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0151] SEQ ID NO:575 (Extracellular domain of human TMEFF2)

[0152] FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0153] TMEFF2 EGF domain SEQ ID NO:577

[0154] HHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCE

[0155] TMEFF2 membrane proximal region SEQ ID NO:629

[0156] NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0157] "TMEFF2-positive cancer" refers to cancer tissue or cancer cells exhibiting measurable levels of TMEFF2 protein. TMEFF2 protein levels can be measured on live or lysed cells using well-known assays such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay. "Overexpression (overexpressed, overexpressing, and overexpressing)" interchangeably refers to a sample, such as cancer cells, malignant cells, or cancer tissue, that has measurably higher levels of tumor antigens compared to a reference sample. Overexpression can be caused by gene amplification or by increased transcription or translation. Protein expression and overexpression in a sample can be measured on live or lysed cells using well-known assays such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay. Polynucleotide expression and overexpression in a sample can be measured, for example, using fluorescence in situ hybridization, Western blotting, or PCR. A protein or polynucleotide is overexpressed when the protein or polynucleotide level in a sample is at least 1.5 times that in a reference sample. The selection of a reference sample is well-known.

[0158] “Sample” means a collection of similar fluids, cells, or tissues separated from a subject, as well as fluids, cells, or tissues present within the subject’s body. Exemplary samples include biological fluids such as blood, serum, and serous fluids, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids from the pleura, pericardium, peritoneum, abdominal cavity, and other body cavities, fluids collected by bronchoalveolar lavage fluid, liquid solutions in contact with the subject or biological sources such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, and tumor tissue removed by fine-needle aspiration or surgical resection.

[0159] As used herein, “cancer cell” or “tumor cell” refers to cancerous, precancerous, or transformed cells in vivo, in vitro, or in tissue culture that exhibit spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. While transformation can occur through infection with transforming viruses and the binding of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, resulting in mutations in endogenous genes. Examples of transformation / cancer include morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignancy, regulation of tumor-specific marker levels, invasion, and tumor growth in suitable animal hosts such as nude mice, in vitro, in vivo, and in vitro (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd edition, 1994)).

[0160] Unless otherwise stated, any numerical value, such as concentrations or concentration ranges described herein, should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include ±10% of the stated value. 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 explicitly include all possible subranges, all individual numerical values ​​within that range, including integers and fractions within such ranges.

[0161] "Effective antigens" are antigens derived from cells of the immune system that can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, and / or cytokine release. Such effector antigens originate from, for example, but not limited to, T cells and natural killer (NK) cells. Examples of suitable specificity against effector antigens include, but are not limited to, CD3 or CD3 subunits, such as CD3ε against T cells and CD16 against NK cells. Such cell surface molecules of effector cells are suitable for mediating cell killing. Effector cells are cells of the immune system that can stimulate or trigger 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, and NCR for NK cells; CD18, CD64, and CD89 for granulocytes; CD18, CD32, CD64, CD89, and mannose receptors for monocytes and macrophages; CD64 and mannose receptors for dendritic cells; and CD35. In some embodiments of the invention, those specificities of effector cells, i.e., cell surface molecules, are adapted to mediate cell killing, thereby inducing cell lysis or apoptosis, when bispecific or multispecific molecules bind to such cell surface molecules.

[0162] A "bispecific CD3 antibody" is a molecule containing 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 containing 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. Bispecific CD3 antibodies can be monovalent in terms of their binding to CD3 or a secondary antigen. In some embodiments, the secondary antigen or target antigen is a cell surface antigen expressed on target cells other than immune effector cells. In some embodiments, the secondary antigen is a tumor-associated antigen (TAA). Exemplary TAAs include PSMA, CD33, IL1RAP, and TMEFF2.

[0163] "Bispecific PSMA×CD3 antibody," "PSMA / CD3 antibody," "bispecific anti-PSMA×CD3 antibody," or "anti-PSMA / CD3 antibody," etc., refer to molecules containing at least one binding domain that specifically binds PSMA and at least one binding domain that specifically binds CD3. The domains that specifically bind PSMA and CD3 are typically V... H / V L Yes. Bispecific anti-PSMA×CD3 antibodies can be monovalent in terms of their binding to PSMA or CD3.

[0164] Terms such as "bispecific CD33×CD3 antibody," "CD33 / CD3 antibody," "bispecific anti-CD33×CD3 antibody," or "anti-CD33 / CD3 antibody" refer to molecules containing 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. Bispecific anti-CD33×CD3 antibodies can be monovalent in terms of their binding to CD33 or CD3.

[0165] Terms such as "bispecific IL1RAP×CD3 antibody," "IL1RAP / CD3 antibody," "bispecific anti-IL1RAP×CD3 antibody," or "anti-IL1RAP / CD3 antibody" refer to molecules containing 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. Bispecific anti-IL1RAP×CD3 antibodies can be monovalent in terms of their binding to IL1RAP or CD3.

[0166] "Bispecific anti-TMEFF2 / anti-CD3 antibody", "TMEFF2 / CD3 antibody", "TMEFF2xCD3 antibody", etc. refer to antibodies that bind to TMEFF2 and CD3.

[0167] "Valence" refers to the specified number of binding sites in a molecule that are specific to the antigen. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites in a molecule, respectively. "Multivalent" refers to the presence of two or more binding sites in a molecule that are specific to the antigen.

[0168] "Antigen-specific CD4+ or CD8+ T cells" refers to CD4+ or CD8+ T cells activated by specific antigens or their immune stimulatory epitopes.

[0169] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably.

[0170] Unless otherwise expressly stated, throughout the specification, the amino acid residues in the constant region of the antibody are described according to the EU index number, as in Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0171] Table 1. Common single-letter and three-letter amino acid codes used in this article

[0172] amino acids 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

[0173] Composition of matter

[0174] This invention provides anti-CD3 antibodies and their antigen-binding fragments, antibodies specifically binding to PSMA and their antigen-binding fragments, antibodies specifically binding to CD33 and their antigen-binding fragments, antibodies specifically binding to IL1RAP and their antigen-binding fragments, 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. This invention also provides polypeptides and polynucleotides or their complementary nucleic acids encoding the antibodies of this invention, vectors, host cells, and methods for their preparation and use.

[0175] General aspects of antibodies described in this article

[0176] The anti-CD3 antibody 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 this invention, unless otherwise stated, the following symbols are used to describe mutations: i) an amino acid substitution at a given position is written as, for example, K409R, which means that lysine at position 409 is replaced by arginine; and ii) for a particular variant, a specific three-letter or single-letter code (including codes Xaa and X) is used to indicate any amino acid residue. Thus, arginine replacing lysine at position 409 is represented as K409R, or any amino acid residue replacing lysine at position 409 is represented as K409X. In the case of lysine deletion at position 409, it is represented by K409*. Those skilled in the art can prepare variants having one or more amino acid substitutions, deletions, or additions.

[0177] These variants may include: (a) variants in which one or more amino acid residues are replaced by conserved or non-conserved amino acids; (b) variants in which one or more amino acids are added to or removed from the polypeptide; (c) variants in which one or more amino acids include substituents; and (d) variants in which the polypeptide is fused with another peptide or polypeptide (such as a fusion coupler, protein tag, or other chemical moiety) to impart useful properties to the polypeptide, such as, for example, antibody epitopes, multihistidine sequences, biotin moieties, etc. The antibody or antigen-binding fragments described herein may include variants in which amino acid residues from one species are replaced at conserved or non-conserved positions with corresponding residues from another species. In other embodiments, amino acid residues at non-conserved positions are replaced by conserved or non-conserved residues. Techniques for obtaining these variants, including genetic techniques (deletion, mutation, etc.), chemical techniques, and enzymatic techniques, are known to those skilled in the art.

[0178] The antibody or antigen-binding fragment described herein may be an IgM, IgD, IgG, IgA, or IgE isotype. In some embodiments, the antibody isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is an IgG1 isotype. In some embodiments, the antibody is an IgG2 isotype. In some embodiments, the antibody is an IgG3 isotype. In some embodiments, the antibody is an IgG4 isotype. The specific portion of an antibody or its antigen-binding fragment is determined by the amino acid sequence and arrangement of the CDR. Therefore, a CDR of one isotype can be converted to another isotype without changing the antigen specificity. Therefore, such antibody isotypes fall within the range of the antibody or antigen-binding fragment described herein.

[0179] Human IgGs are classified 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 exhibit major differences 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 cell 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), leading to the lysis of the target cell. In CDC, the antibody mediates the killing of target cells by triggering a complement cascade reaction on the cell surface. In ADCP, the antibody mediates the elimination of antibody-coated target cells through internalization by phagocytes (such as macrophages or dendritic cells). The antibodies described herein include antibodies having the aforementioned features of variable domains combined with any IgG isotype, including modified forms in which the Fc region has been modified to modulate various effector functions.

[0180] For many applications of therapeutic antibodies, Fc-mediated effector functions are undesirable because they may pose a potential safety risk due to cellular depletion. Modification of 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 first component of activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb), FcγR, or complement (C1q) components depends on residues located in the hinge region and CH2 domain. Mutations can be introduced into IgG1, IgG2, and IgG4 to reduce or silence Fc-mediated effector functions. Antibodies described herein may include these modifications.

[0181] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibody comprises an engineered Fc region having one or more of the following properties: (a) reduced effector function when compared with 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.

[0182] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are, for example, IgG1, IgG2, IgG3, or IgG4 isotypes. In some embodiments where the antibody is of the 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 is of the IgG1 isotype, the antibody contains L234A and L235A substitutions in its Fc region. The antibodies described herein may include these modified forms.

[0183] In some implementations, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are IgG4 isotypes and optionally include heavy chain substitution S228P.

[0184] In some implementations, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are IgG1 isotypes that, when compared with wild-type IgG1, optionally include heavy chain substitutions L234A, G237A, P238S, H268A, A330S, and P331S.

[0185] In some implementations, the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are IgG2 isotypes that, when compared with wild-type IgG2, optionally include heavy chain substitutions L234A, G237A, P238S, H268A, V309L, A330S, and P331S.

[0186] Wild-type IgG4

[0187] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:602)

[0188] Wild-type IgG1

[0189] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0190] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0191] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:601)

[0192] Wild-type IgG2

[0193] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0194] GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:711)

[0195] In some embodiments, labeled anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies are provided. Exemplary labels or portions for direct detection (e.g., fluorescent labels, chromogenic labels, high-density electron labels, chemiluminescent labels, and radioactive labels) and labels and portions (e.g., enzymes or ligands) for indirect detection (e.g., via enzyme reactions or molecular interactions) are also provided. Exemplary labels include radioactive labels (e.g., 32 P, 14 C 111 I, 125 I, 3 H, 131 I) Fluorescent labels (such as...) 649), epitope tags, biotin, chromophore markers, ECL markers, or enzymes. More specifically, the markers include ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and β-galactosidase, polyhistidine (HIS tag), acridine dyes, anthocyanin dyes, fluorescent ketone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Dyes, etc.

[0196] In addition to the anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies and antigen-binding fragments described herein, a polynucleotide sequence encoding the antibody and antigen-binding fragment is also provided. A vector comprising the polynucleotide is 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 vector are also described. These cells may 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 antibody may also be produced by hybridoma cells.

[0197] Production of monospecific antibodies

[0198] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2 and / or anti-IL1RAP antibodies of the present invention are human.

[0199] In some embodiments, the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2 and / or anti-IL1RAP antibodies of the present invention are humanized.

[0200] The monospecific antibodies of the present invention described herein (e.g., anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies) can be generated using various 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, mice or other host animals (such as hamsters, rats, or monkeys) are immunized with human, chimpanzee, or macaque PSMA, CD33, IL1RAP, TMEFF2, or CD3, or fragments of PSMA, CD33, IL1RAP, TMEFF2, or CD3 (such as the extracellular domains of PSMA, CD33, IL1RAP, TMEFF2, or CD3). Splenocytes from the immunized animal are then 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 a single immortalized hybridoma cell are screened for use in the preparation of antibodies with desired properties (such as binding specificity, cross-reactivity, or lack thereof, as well as affinity for the antigen).

[0201] Various host animals can be used to generate the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies of the present invention. For example, Balb / c mice can be used to generate mouse anti-human PSMA antibodies. Various techniques can be used to humanize antibodies prepared from Balb / c mice and other non-human animals to produce more human-like sequences.

[0202] Exemplary humanization techniques, including the selection of human receptor frameworks, are known and include CDR grafting (US Patent 5,225,539), SDR grafting (US Patent 6,818,749), surface remodeling (Padlan, 1991, Mol Immunol, Vol. 28, pp. 489-499), surface remodeling of specificity-determining residues (US Patent Publication 2010 / 0261620), human framework modification (US Patent 8,748,356), or hyperhumanization (US Patent 7,709,226). In these methods, the CDR of the parent antibody is transferred to a human framework, which can be selected based on its overall homology with the parent framework, similarity of CDR length, or canonical structural identity, or a combination thereof.

[0203] Humanized antibodies can be further optimized to improve their selectivity or affinity for desired antigens by: introducing modified framework support residues to maintain binding affinity (reverse mutation) by employing techniques such as those described in international patent publications WO1090 / 007861 and WO1992 / 22653, or by introducing variants at any CDR to improve antibody affinity.

[0204] 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 documented in, for example, US 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 & Jakob ovits, 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 The method is described in *Immunol Methods*, Vol. 231, pp. 11–23; Yang et al., 1999, *Cancer Res*, Vol. 59, pp. 1236–1243; and Brüggemann and Taussig, 1997, *Curr Opin Biotechnol*, Vol. 8, pp. 455–458. Endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal genome via homologous or non-homologous recombination using transchromosomal or microgenetic methods. Companies such as Regeneron (http: / / www.regeneron.com), HarbourAntibodies (http: / / www.harbourantibodies.com), OpenMonoclonal 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 technology to provide human antibodies against selected antigens.

[0205] Human antibodies can be selected from phage display libraries in which phages are engineered to express human immunoglobulins or portions thereof, such as Fab, single-chain antibodies (scFv), or variable regions of unpaired or paired antibodies (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 phage display libraries that express the variable regions of the antibody heavy and light chains as fusion proteins 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 PSMA, CD33, IL1RAP, TMEFF2, or CD3 in humans and / or cynomolgus monkeys 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.

[0206] The preparation of immunogenic antigens and the generation of monoclonal antibodies can be performed using any suitable technique, such as recombinant protein production. Immunogenic antigens can be administered to animals in the form of purified proteins or mixtures of proteins (including whole cells, cell extracts, or tissue extracts), or the antigens can be de novo formed in animals from nucleic acids encoding said antigens or portions thereof.

[0207] The generation and use of bispecific and multispecific CD3 antibodies

[0208] This invention provides bispecific and multispecific antibodies. The bispecific antibody comprises a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. The second antigen may be a tumor-associated antigen (TAA) or an antigen on pathogenic cells.

[0209] Exemplary anti-CD3 antibodies that can be used to engineer bispecific and multispecific antibodies comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen include CD3 antibodies comprising the V shown in Tables 7A and 7B. H / V L The sequences and heavy / light chain CDRs, as well as their engineered variants described in Tables 10 and 11 and the accompanying text, are provided. 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 to CD3 and a second domain that specifically binds to a second antigen.

[0210] The anti-CD3 antibody CDR and / or VH / VL domains described herein can be incorporated into bispecific antibodies containing a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen.

[0211] The anti-CD3 antibody CDR and / or VH / VL domains described herein can be incorporated into bispecific antibodies containing the PSMA-binding VH / VL domains described herein and in Table 23. The anti-CD3 antibody and / or VH / VL domains described herein can be incorporated into bispecific antibodies containing the IL1RAP-binding VH / VL domains described herein and in Table 30. The anti-CD3 antibody CDR and / or VH / VL domains described herein can be incorporated into bispecific antibodies containing the CD33-binding VH / VL domains described herein and in Table 38. For example, the VH / VL domains of 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.

[0212] Exemplary TAAs are PSMA, CD33, TMEFF2, and IL1RAP. The exemplary multispecific PSMA×CD3, CD33×CD3, TMEFF2×CD3, and IL1RAP×CD3 antibodies provided herein have a first domain that specifically binds to CD3 and a second domain that specifically binds to PSMA, CD33, TMEFF2, or IL1RAP. Exemplary anti-PSMA antibodies that can be used to engineer bispecific PSMA×CD3 molecules are those described herein, and may contain 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 may contain 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 may contain 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, which may contain heavy chain and light chain variable region sequences, including but not limited to the heavy chain and light chain variable region sequences provided in Tables 59 and 66-68.

[0213] The binding of the generated bispecific antibody to CD3 and / or the second antigen and / or its desired functional characteristics, such as T cell-mediated killing of cells expressing the second antigen, can be tested.

[0214] The bispecific antibodies provided in this article include antibodies with full-length antibody structures.

[0215] A "Fab arm" or "half-molecule" refers to a heavy-light chain pair that specifically binds to an antigen.

[0216] As described herein, the full-length bispecific antibody can be generated, for example, through Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies, by introducing a substitution at the heavy chain CH3 junction 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 isomerization and CH3 domain dissociation-association. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The resulting free cysteine ​​residue of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed through dissociation-association. The CH3 domain of the Fab arm can be modified to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding a different epitope: an epitope on CD3 and an epitope on the second antigen.

[0217] "Homodimerization" refers to the interaction between two heavy chains that have the same CH3 amino acid sequence. "Homodimer" refers to an antibody with two heavy chains containing the same CH3 amino acid sequence.

[0218] Heterodimerization refers to the interaction between two heavy chains with different CH3 amino acid sequences. A heterodimer is an antibody containing two heavy chains with different CH3 amino acid sequences.

[0219] In some implementations, bispecific antibodies include those such as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Button (Genentech), CrossMAbs (Roche), and electrostatic pairings (Chugai, Amgen, Novo Nordisk, Oncomed), LUZ-Y (Genentech), chain exchange engineered domain bodies (SEEDbody) (EMDSerono), Biclonic (Merus), and Design of the technology (Genmab A / S).

[0220] The Triomab quadroma technology can be used to generate full-length bispecific antibodies that incorporate the VH and VL regions of the anti-CD3 antibody of this invention. The Triomab technology facilitates Fab arm exchange between two parental chimeric antibodies, one parental mAb having IgG2a and the second parental mAb having the rat IgG2b constant region, thereby generating chimeric bispecific antibodies.

[0221] The “button” strategy (see, for example, international publication WO2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the CH3 domain boundaries in human IgG can be mutated at positions affecting CH3 domain interactions, thereby promoting heterodimer formation. Amino acids with small side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to the first antigen, and amino acids with large side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction between the heavy chains with “buttons” and those with “buttons.” Exemplary CH3 substitution pairs forming buttons and clasps (represented as modification positions in the first CH3 domain of the first heavy chain / modification positions 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.

[0222] CrossMAb technology can be used to generate full-length bispecific antibodies. In addition to facilitating Fab wall exchange using a “button” strategy, CrossMAb has exchanged CH1 and CL domains in one of its halves to ensure proper light chain pairing of the resulting bispecific antibody (see, for example, U.S. Patent 8,242,247).

[0223] Other exchange strategies can be used to generate full-length bispecific antibodies by exchanging variable or constant domains, or both, between or within the heavy chain and the light chain in one or both arms of the bispecific antibody. These exchanges include, for example, VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1, as described in international patent publications WO2009 / 080254, WO2009 / 080251, WO2009 / 018386, and WO2009 / 080252.

[0224] Other strategies can also be used, such as promoting heterodimerization of heavy chains by electrostatic interactions through the substitution of positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in U.S. Patent Publications US2010 / 0015133, US2009 / 0182127, US2010 / 028637, or US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (represented as the modification position in the first CH3 domain of the first heavy chain / the modification position 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_Y 407A / 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.

[0225] The LUZ-Y technique can be used to generate bispecific antibodies. In this technique, a leucine zipper is added to the C-terminus of the CH3 domain to drive the assembly of a heterodimer from a parent mAb, which is subsequently purified and removed, as described in Wranik et al., 2012, J Biol Chem, Vol. 287, No. 52, pp. 42221-42229.

[0226] SEEDbody technology can be used to generate bispecific antibodies. SEEDbodies have selected IgG residues substituted with IgA residues in their constant domain to facilitate heterodimerization, as described in U.S. Patent US20070287170.

[0227] According to the method described in International Patent Publication WO2011 / 131746, the bispecific antibody described herein can be produced in vitro in a cell-free environment by introducing an asymmetric mutation into the CH3 region of two monospecific homodimers and forming a bispecific heterodimer from the two parental monospecific homodimers under reducing conditions that allow disulfide isomerization. In these methods, a first monospecific bivalent antibody (i.e., an antibody that specifically binds to a second antigen; e.g., anti-PSMA, anti-CD33, anti-TMEFF2, and / or anti-IL1RAP antibodies) 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; these antibodies are incubated together under reducing conditions sufficient to cause disulfide isomerization of cysteine ​​residues in the hinge region; thereby generating a bispecific antibody via Fab arm exchange. The incubation conditions are then 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, incubation for at least 90 minutes 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, at a temperature of at least 20°C.

[0228] In some embodiments described herein, a bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen contains at least one substitution in the constant domain of the antibody CH3.

[0229] In some embodiments described herein, at least one substitution in the constant domain of the antibody CH3 is a K409R, F405L, or a F405L and R409K substitution, wherein the residues are numbered according to the EU index.

[0230] Antibody domains and position numbering are well-known. "Asymmetry" refers to the different substitutions within the two CH3 domains of the two independent heavy chains of an antibody. The IgG1 CH3 region is typically composed of residues 341-446 on IgG1 (residues are numbered according to the EU index).

[0231] In some embodiments described herein, the bispecific antibody is an IgG1 isotype and, when compared with wild-type IgG1, contains an F405L substitution in the first heavy chain (HC1) and a K409R substitution in the second heavy chain (HC2).

[0232] In some embodiments described herein, the bispecific antibody is an IgG1 isotype and, when compared with wild-type IgG1, contains a K409R substitution in the first heavy chain (HC1) and an F405L substitution in the second heavy chain (HC2).

[0233] In some embodiments described herein, the bispecific antibody is an IgG4 isotype, and when compared with wild-type IgG4, it contains an S228P substitution in HC1 and an S228P, F405L, and R409K substitution in HC2.

[0234] In some embodiments described herein, the bispecific antibody is an IgG4 isotype, and when compared with wild-type IgG4, it includes S228P, F405L and R409K substitutions in HC1 and S228P substitution in HC2.

[0235] In some embodiments described herein, the bispecific antibody is an IgG4 isotype, and when compared with wild-type IgG4, it includes S228P, F234A, and L235A substitutions in HC1 and S228P, F234A, L235A, F405L, and R409K substitutions in HC2.

[0236] In some embodiments described herein, the bispecific antibody is an IgG4 isotype, and when compared with wild-type IgG4, it includes S228P, F234A, L235A, F405L and R409K substitutions in HC1 and S228P, F234A and L235A substitutions in HC2.

[0237] In some embodiments described herein, when the residues are numbered according to the EU index, the bispecific antibody of the present invention contains at residue positions 350, 366, 368, 370, 399, 405, 407 or 409 in HC1 and HC2, at at least one, two, three, four, five, six, seven or eight asymmetric substitutions.

[0238] In some embodiments described herein, when the residues are numbered according to the EU index, the bispecific antibody of the present invention contains at residue positions 350, 370, 405 or 409 in HC1 and HC2, at least one, two, three or four asymmetric substitutions.

[0239] In some embodiments described herein, when the residues are numbered according to the EU index, the bispecific antibody of the present invention contains at least one asymmetric substitution at residue position 405 or 409 in HC1 and HC2.

[0240] Standard methods are typically used to perform substitutions at the DNA level down to the molecular level (such as constant structural domains of antibodies).

[0241] The antibodies of this invention can be engineered into various known antibody forms.

[0242] In some embodiments, the bispecific antibody of the present invention is a cross-linker.

[0243] In some embodiments, the bispecific antibodies of the present invention include recombinant IgG-like dual-targeting molecules, wherein each flanking the molecule contains Fab fragments or portions of Fab fragments of at least two different antibodies; IgG fusion molecules, wherein a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; Fc fusion molecules, wherein a single-chain Fv molecule or a stable bispecific antibody is fused with a heavy chain constant domain, an Fc region, or a portion thereof; Fab fusion molecules, wherein different Fab fragments are fused together; and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and bispecific antibodies, wherein different single-chain Fv molecules or different bispecific antibodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused with each other or with another protein or carrier molecule.

[0244] In some implementations, recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK / Domantis), dual-antibody (Genentech), and mAb2 (F-Star).

[0245] In some implementations, the IgG fusion molecule includes dual variable domain (DVD)-Ig (Abbott), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (BiogenIdec), and TvAb (Roche).

[0246] In some implementations, Fc fusion molecules include ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), and dual-affinity retargeting technology (Fc-DART) (MacroGenics).

[0247] In some implementations, 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, bivalent antibody domain-based antibodies include bispecific T-cell adaptors (BITE) (Micromet), tandem bivalent antibodies (Tandab) (Affimed), dual-affinity retargeting molecules (DART) (MacroGenics), single-chain bivalent antibodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (EpigenBiotech), dual-targeting nanobodies (Ablynx), and antibodies that target only the heavy chain domain. 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 Nunz-Prado et al., 2015, Drug Discovery Today, Vol. 20, No. 5, pp. 588-594.

[0248] Table 2 summarizes the exemplary monoclonal antibodies described herein that can be used to generate the bispecific antibodies of the present invention.

[0249] Table 2. Exemplary monoclonal antibodies that can be used to generate the bispecific antibodies of the present invention.

[0250]

[0251] Exemplary anti-TMEFF2 antibodies that can be used to engineer bispecific TMEFF2×CD3 molecules are those described herein, which may contain heavy chain and light chain variable region sequences, including but not limited to the heavy chain and light chain variable region sequences provided in Tables 59 and 66-68.

[0252] In some embodiments, the anti-CD3 antibody is a multispecific antibody, such as a bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to 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 include PSMA, CD33, TMEFF2, and IL1RAP.

[0253] The present invention provides a bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen.

[0254] In some embodiments, the first structural domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, as specified in SEQ ID NO: 662, 663, 664, 671, 673, and 690. In some embodiments, the first structural domain comprises VH and VL, respectively, as specified in SEQ ID NO: 652 and 661. In some embodiments, the first structural domain comprises HC and LC, respectively, as specified in SEQ ID NO: 640 and 676. In some embodiments, the first structural domain comprises VH and VL, respectively, as specified in SEQ ID NO: 657 and 678. In some embodiments, the first structural domain comprises HC and LC, respectively, as specified in SEQ ID NO: 675 and 677.

[0255] In some embodiments, the first structural domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, as specified in SEQ ID NO: 662, 663, 664, 773, 673, and 690. In some embodiments, the first structural domain comprises VH and VL, respectively, as specified in SEQ ID NO: 657 and 678. In some embodiments, the first structural domain comprises HC and LC, respectively, as specified in SEQ ID NO: 675 and 677.

[0256] The present invention also provides a bispecific antibody for use in treatment, the bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen of the present invention.

[0257] The present invention also provides a bispecific antibody for use in the treatment of cell proliferation disorders, the bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen of the present invention.

[0258] The present invention also provides a bispecific antibody for use in the treatment of cancer, the bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen of the present invention.

[0259] The present invention also provides a bispecific antibody for use in the treatment of autoimmune diseases, the bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen of the present invention.

[0260] 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 to CD3 and a second domain that specifically binds to a second antigen of the present invention.

[0261] The present invention also provides a bispecific antibody for use in the preparation of a medicament for treating autoimmune diseases, the bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen of the present invention.

[0262] Another aspect of the invention is a method of treating a subject with a proliferative or autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of the anti-CD3 antibody of the invention. In some embodiments, the anti-CD3 antibody or a bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the 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 a bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the 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 a bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the invention is administered to the subject at a dose of about 1 mg / kg. In some embodiments, the anti-CD3 antibody or a bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the present invention is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. In some embodiments, the anti-CD3 antibody or a bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the present invention is administered subcutaneously. In some embodiments, the anti-CD3 antibody or a bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the present invention is administered intravenously.

[0263] 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 bowel cancer, large bowel 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 prelymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassified diffuse red pulp small B-cell lymphoma of the spleen, variant hairy cell leukemia, Waldenström macroglobulinemia, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, mucosa-associated lymphoid tissue extranodal marginal zone lymphoma (MALT lymphoma), lymph node marginal zone lymphoma, pediatric lymph node marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicular center lymphoma, T-cell / histocyte-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-associated multicentric Castleman disease, primary exudative lymphoma, unclassified B-cell lymphoma with characteristics between diffuse large B-cell lymphoma and Burkitt lymphoma, and unclassified B-cell lymphoma with characteristics between diffuse large B-cell lymphoma, classical Hodgkin lymphoma, and light chain amyloidosis.

[0264] In some implementations, the cancer is esophageal cancer. In other implementations, the cancer is adenocarcinoma, such as metastatic adenocarcinoma (e.g., colorectal adenocarcinoma, gastric adenocarcinoma, or pancreatic cancer).

[0265] 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 thrombocytopenic purpura, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, Raynaud's syndrome, Sjögren's syndrome, glomerulonephritis, neuromyelitis optica (NMO), and IgG neuropathy.

[0266] In another aspect, the present invention features a kit comprising: (a) a composition comprising either the aforementioned anti-CD3 antibody or a bispecific antibody, the bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the present invention; and (b) instructions for use, including instructions for administering the composition to a subject to treat or delay the progression of a proliferative disease. The term "instructions for use" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, containing information regarding indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings related to the use of such therapeutic products.

[0267] In any of the foregoing uses or methods, the subject may be a human being.

[0268] Polynucleotides, vectors and host cells

[0269] Also disclosed are isolated polynucleotides encoding the anti-CD3 antibody or the bispecific antibody of the present invention, the bispecific antibody comprising a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen. Isolated polynucleotides capable of encoding the variable domains provided herein may be included on the same or different vectors to generate the antibody or antigen-binding fragment of the present invention.

[0270] In some embodiments, the polynucleotides of the present invention comprise a polynucleotide encoding a leader sequence. Any leader sequence known in the art may be used. The polynucleotide encoding the leader sequence may comprise a restriction endonuclease cleavage site or a translation initiation site.

[0271] Vectors containing the polynucleotides of the present invention are also provided. The vector may be an expression vector. The expression vector may 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 various host cells are well known and include, but are not limited to, plasmids, phage particles, phages, baculoviruses, baculosomes, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0272] Recombinant expression vectors within the scope of this specification include synthetic or cDNA-derived nucleic acid fragments that encode at least one recombinant protein operatively linked to a suitable regulatory element. Such regulatory elements may include a transcription promoter, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. Expression vectors, particularly mammalian expression vectors, may also include one or more non-transcriptional elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcriptional sequences, 5' or 3' untranslated sequences (such as an essential ribosome binding site), polyadenylation sites, splicing donor and acceptor sites, or transcription termination sequences. An origin of replication conferring the ability to replicate in the host may also be incorporated.

[0273] The transcriptional and translational control sequences in the expression vector used for transforming vertebrate cells can be provided by a viral source. Exemplary vectors include Okayama and Berg, 3Mol.Cell.Biol.280 (1983).

[0274] In some embodiments, the antibody-coding sequence or the antigen-binding fragment-coding sequence is placed under the control of a potent constitutive promoter, such as promoters for genes including hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin, human myosin, human hemoglobin, human muscle creatine, etc. Furthermore, many viral promoters constitutively function in eukaryotic cells and are suitable for use with the embodiments described above. These viral promoters include, but are not limited to, the immediate early promoter of cytomegalovirus (CMV), early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) sequence of Maronnie leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Raul's sarcoma virus (RSV), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. In one embodiment, the coding sequence of a PSMA-specific antibody or its antigen-binding fragment is placed under the control of an inducible promoter (such as a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, or a promoter containing one or more interferon-stimulated response elements (ISREs) (such as protein kinase R2',5'-oligoadenylate synthase, the Mx gene, ADAR1, etc.).

[0275] The vectors described herein may contain one or more internal ribosome entry sites (IRES). The inclusion of IRES sequences in fusion vectors may enhance 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 aforementioned nucleic acid sequences. Vector components may be sequentially linked, arranged in a manner that provides optimal spacing for gene product expression (i.e., by introducing “spacer” nucleotides between ORFs), or positioned in another manner. Regulatory elements such as IRES motifs may also be arranged to provide optimal spacing for expression.

[0276] The vector may contain selection markers well known in the art. Selection markers include positive and negative selection markers, such as antibiotic resistance genes (e.g., neomycin resistance genes, hygromycin resistance genes, kanamycin resistance genes, tetracycline resistance genes, penicillin resistance genes), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence encoding the selection marker or cloning site may be upstream or downstream of the nucleic acid sequence encoding the polypeptide of interest or the cloning site.

[0277] The vectors described herein can be used to transform various cells with genes encoding the antibodies or antigen-binding fragments. For example, the vectors can be used to produce anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibodies, or to produce cells that generate antigen-binding fragments. Therefore, the present invention also provides a host cell comprising the vector of the present invention.

[0278] Various techniques for introducing foreign genes into cells are known in the art, and for the purpose of implementing the methods described herein, these techniques can be used to construct recombinant cells according to various embodiments described and illustrated herein. The techniques used should enable the stable transfer of the heterologous gene sequence to the host cell such that the heterologous gene sequence is heritable and can be expressed by cell daughter cells, thereby preserving the necessary development and physiological functions of the recipient cell. 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, protoplast 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). Cell transformation can also be achieved using calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells.

[0279] Cells suitable for expression of the antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably plant, rodent, or human-derived cells, 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 the BHK cell line. Furthermore, hybridoma cells can be used to express the antibody. Methods for generating hybridomas are well-established in the art.

[0280] Cells transformed with the expression vector 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 for subclones exhibiting the desired phenotype (such as high-level expression, enhanced growth properties, or the ability to produce proteins with the desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications). These phenotypes may be due to the inherent properties of a given subclone or due to mutations. Mutations can be achieved using chemicals, UV wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

[0281] Pharmaceutical composition / application

[0282] This 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 into a pharmaceutical composition containing an effective amount of the antibody as the active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or medium with which the antibody of the present invention is administered. Such mediators can be liquids, such as water and oils, including oils derived from petroleum, animals, plants, or synthetic oils, 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 generally free of particulate matter. They can be sterilized using conventional, known sterilization techniques (e.g., filtration). The composition may contain, as needed, pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants, etc. In such pharmaceutical formulations, the concentration of the antibody of the present invention can vary from less than about 0.5% by weight, typically to at least about 1% to as much as 15 or 20%, and can be selected based primarily on the desired dose, fluid volume, viscosity, etc., depending on the specific method of administration chosen. Suitable media and formulations containing other human proteins (e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, edited by Troy, DB, Lipincott Williams and Wilkins, Philadelphia, PA, 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092 (see especially pp. 958-989).

[0283] The therapeutic use of the antibodies for the purposes of this invention can be carried out via any suitable route of delivery to the host, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, nasal, vaginal, rectal), using formulations in tablet, capsule, solution, powder, gel, or granule form; and contained in syringes, implantation devices, osmotic pumps, boxes, micropumps; or other methods well known in the art as understood by a person skilled in the art. Site-specific administration can be achieved, for example, by intratumoral, parenteral, bronchial, abdominal, cystic, cartilage, cavity, body cavity, cerebellum, ventricle, colon, cervical canal, stomach, liver, heart, bone, pelvis, pericardium, peritoneum, pleura, prostate, lung, rectum, kidney, retina, spine, synovium, chest, uterus, blood vessel, bladder, lesion, vagina, rectum, oral cavity, sublingual, nasal, or percutaneous delivery.

[0284] The antibodies of this invention can be administered to subjects via any suitable route, such as intravenous (iv) infusion or bolus injection, intramuscular or subcutaneous or intraperitoneal parenteral administration. Intravenous infusion can be administered 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.

[0285] The dose administered to the subject is sufficient to alleviate or at least partially halt the disease being treated (“therapeutic effective dose”), and may sometimes be from 0.005 mg / kg to about 100 mg / kg, for example from about 0.05 mg / kg to about 30 mg / kg, or from 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, etc. / kg, 8mg / kg, 9mg / kg or 10mg / kg, but may be even higher, such as about 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70mg / kg, 80mg / kg, 90mg / kg or 100mg / kg.

[0286] Fixed unit doses, such as 50 mg, 100 mg, 200 mg, 500 mg, or 1000 mg, can also be administered, 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. Patients are typically treated with doses between 1 and 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 times), but doses of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more may be administered.

[0287] The antibody of the present invention can be repeated after one, two, three, four, five, six, 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 periods. The treatment process can also be repeated, as with chronic administration. Repeated administration can be at the same dose or different doses. For example, the antibody of the present invention described herein can be administered intravenously at a weekly interval of 8 mg / kg or 16 mg / kg for 8 weeks, followed by administration at a weekly interval of 8 mg / kg or 16 mg / kg for another 16 weeks, and then administration at a weekly interval of 8 mg / kg or 16 mg / kg for another 4 weeks.

[0288] For example, the antibody described herein may be present at at least one of the following dates: 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 days after the start of treatment, or alternatively, at least one of the following dates: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the start of treatment. Or any combination thereof, used as a single dose or divided doses every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof, in an amount of about 0.1-100 mg / kg as a daily dose, 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.

[0289] The antibodies described in this article 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.

[0290] The antibodies described herein can be stored by low-pressure lyophilization and reconstituted in a suitable carrier before use. This technique has been proven effective for routine protein preparations and can be performed using well-known lyophilization and reconstitution techniques.

[0291] Methods for detecting CD3, PSMA, CD33, IL1RAP, TMEFF2 or target antigens and CD3

[0292] This article provides a method for detecting CD3, PSMA, CD33, TMEFF2 or IL1RAP in a sample. The method includes obtaining a sample, contacting the sample with the anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2 or anti-IL1RAP antibody of the present invention, and detecting the antibody that binds to CD3, PSMA, CD33, TMEFF2 or IL1RAP in the sample.

[0293] A method for detecting CD3 and a second antigen (e.g., PSMA, CD33, TMEFF2, or IL1RAP) in a sample is also provided, the method comprising obtaining a sample, contacting the sample with a bispecific antibody comprising a first domain specifically binding CD3 and a second domain specifically binding a second antigen, and detecting the antibody binding CD3 and the second antigen in the sample.

[0294] In some of the embodiments described herein, the samples may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically removed tumor tissue, biopsies, including fine-needle aspiration tissue), histological preparations, etc.

[0295] The antibodies of the present invention can be detected using known methods. Exemplary methods include directly labeling the antibodies using fluorescent or chemiluminescent labels, or radiolabels, or attaching an easily detectable portion (such as biotin, enzyme, or epitope tag) to the antibodies of the present invention. Exemplary labels and portions are ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and β-galactosidase, polyhistidine (HIS tag), acridine dyes, anthocyanin dyes, fluorescent ketone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes and dye.

[0296] The antibodies described herein can be used in a variety of assays to detect CD3, PSMA, CD33, IL1RAP, TMEFF2, or CD3 and secondary antigens in samples. Exemplary assays include Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or ELISA.

[0297] Antibody kit

[0298] This invention also provides a kit comprising one or more of anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibodies or bispecific antibodies, wherein the bispecific antibody comprises a first domain specifically binding to CD3 and a second domain specifically binding to a second antigen of the present invention or an antigen-binding fragment thereof. The kit can be used to implement methods using anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibodies or bispecific antibodies comprising a first domain specifically binding to CD3 and a second domain specifically binding to 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. Therefore, the kit may include one or more of the following: antibodies or antigen-binding fragments described herein, and containers for containing the antibodies or fragments when not in use, instructions for use of the antibodies or fragments, antibodies or fragments attached to a solid support, and / or antibodies or fragments in detectable labeled forms as described herein.

[0299] This invention also provides kits containing antibodies that specifically bind PSMA as described herein. This invention also provides kits containing antibodies that specifically bind CD33 as described herein. This invention also provides kits containing antibodies that specifically bind IL1RAP as described herein. This invention also provides kits containing antibodies that specifically bind TMEFF2 as described herein.

[0300] The kit can be used for therapeutic purposes and as a diagnostic kit.

[0301] The kit can be used to detect the presence of CD3, PSMA, CD33, IL1RAP, TMEFF2 and / or secondary antigens in biological samples.

[0302] In some embodiments, the kit comprises the antibody of the present invention described herein and reagents for detecting the antibody. The kit may also include 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 with a label or therapeutic agent, or radiation protection compositions; means or other materials for preparing the antibody for administration; a pharmaceutically acceptable carrier; and means or other materials for administration to a subject.

[0303] In some implementations, the kit includes a container holding the antibody of the present invention and instructions for use of the kit.

[0304] In some implementations, the antibodies in the kit are labeled.

[0305] CD3-specific antibodies

[0306] 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 and fragments thereof of the present invention 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 a 5-fold. In this case, 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. This human / cynomolgus monkey cross-reactivity leads to highly comparable results and significantly increased predictive power in animal studies compared to species-specific alternative molecules. In some embodiments, the anti-CD3 antibodies and fragments thereof of the present invention bind to epitopes formed by CD3e / d subunits. The CD3-specific antibody can be human, humanized, or chimeric. This article also provides examples of human antibodies generated in OmniRat (OpenMonocmatic Technologies (“OMT”), Palo Alto, California, USA, omniab.com).

[0307] In some embodiments described herein, the anti-CD3 antibody or fragment thereof has one, two, three, four, or five of the following characteristics:

[0308] a) Human and cynomolgus monkey CD3+ T lymphocytes bound with a calculated EC50 of 20 nM or less, and cynomolgus monkey CD3-expressing HEK cells bound with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between the bound CD3+ T lymphocytes and the bound cynomolgus monkey CD3-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole-cell binding assay using flow cytometry at 0°C;

[0309] b) With an equilibrium dissociation constant (K) of 12 nM or less. D ) combines with recombinant CD3d from humans (SEQ ID NO:691), or with recombinant CD3e from humans (SEQ ID NO:636), or with recombinant CD3e from cynomolgus monkeys (SEQ ID NO:693), where K D Resonance determination of the ProteOnXPR36 system using Proteon surface plasmon oscillators at +25°C;

[0310] c) If detected by peptide profiling analysis, it shows no methionine or tryptophan oxidation, or no asparagine deamidation, or no asparagine isomerization.

[0311] d) Binding to CD3e residues 1-6, as determined by X-ray crystallography; or

[0312] e) Activate T cells or induce CD69 expression to a level similar to cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0313] The anti-CD3 antibody and its fragment of the present invention have an in vitro binding affinity (KL) 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. d (e.g., determined by flow cytometry).

[0314] In some aspects, the anti-CD3 antibody and its fragment of the present invention are bivalent antibodies, which have an in vitro binding affinity (KL) 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, and most preferably about 100 nM, for human T cells expressing human CD3. d (e.g., determined by flow cytometry).

[0315] In some aspects, the anti-CD3 antibody and its fragment of the present invention are monovalent constructs having an in vitro binding affinity (K0.05) 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 100 nM to about 250 nM, and most preferably about 250 nM, for human T cells expressing human CD3.d (e.g., determined by flow cytometry).

[0316] In one aspect, the anti-CD3 antibody and its fragment described herein competitively bind to CD3 with the commercially available CD3 antibody SP34-2 (BDBiosciences551916), as determined by a competitive binding assay of primary human T cells using an Alexa Fluor488-conjugated SP34-2 antibody, which was measured using flow cytometry.

[0317] In one respect, anti-CD3 antibodies and their fragments do not exhibit post-translational modifications, including no oxidation, no deamidation, and no aspartic acid isomerization, as determined by peptide profiling analysis.

[0318] In one respect, anti-CD3 antibodies and their fragments effectively activate T cells and induce CD69 expression to a level similar to 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.

[0319] In one aspect, the anti-CD3 antibodies and fragments thereof described herein have a total enthalpy of folding 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 some aspects, the anti-CD3 antibodies and fragments thereof of the present invention have a total folding enthalpy of about 418 kcal / mol, 545 kcal / mol, about 402 kcal / mol or about 406 kcal / mol, and the anti-CD3 antibodies are respectively CD3B376 (IgG4PAA) molecules, CD3B450 (IgG4PAA) molecules, CD3B389 (IgG1σ) molecules and CD3B467 (IgG1σ) molecules.

[0320] Exemplary examples of such antibodies include CD3B311, CD3B312, CD3B313, CD3B314, CD3B315, CD3B316, CD3B317, CD3B334, CD3B376, CD3B389, CD3B450, and CD3B467, as well as CD3B376 and CD3B450 engineered in monovalent form.

[0321] The anti-CD3 antibody or antigen-binding fragment of the present invention may exist in various forms, but will include one or more of the antibody variable domain fragments or CDRs shown in Table 7A and their engineered variants, such as those shown or described in Tables 9 and 10, and their accompanying descriptions.

[0322] The present invention also provides anti-CD3 antibodies or antigen-binding fragments thereof, comprising heavy chains of HCDR1, HCDR2, and HCDR3 containing any of the antibodies described in Table 7B. The present invention also provides anti-CD3 antibodies or antigen-binding fragments thereof, comprising heavy chains of HCDR1, HCDR2, and HCDR3 containing any of the antibodies described in Table 7B, and light chains of LCDR1, LCDR2, and LCDR3 containing any of the antibodies described in Table 7B. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention compete with antibodies or antigen-binding fragments thereof comprising heavy chains of HCDR1, HCDR2, and HCDR3 containing any of the antibodies described in Table 7B, and light chains of LCDR1, LCDR2, and LCDR3 containing any of the antibodies described in Table 7B for binding to CD3.

[0323] In some embodiments, the anti-CD3 antibody of the present invention or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 contained in 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.

[0324] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises LCDR1, LCDR2 and LCDR3 contained in 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.

[0325] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises:

[0326] HCDR1 of SEQ ID NO: 662, 665 or 666;

[0327] HCDR2 of SEQ ID NO: 663, 689 or 695; and

[0328] HCDR3 of SEQ ID NO:664;

[0329] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises:

[0330] LCDR1 of SEQ ID NO: 773, 710, 674 or 671;

[0331] LCDR2 of SEQ ID NO: 669 or 673; and

[0332] LCDR3 of SEQ ID NO:670.

[0333] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises:

[0334] HCDR1 of SEQ ID NO: 662, 665 or 666;

[0335] HCDR2 of SEQ ID NO: 663, 689 or 695;

[0336] HCDR3 of SEQ ID NO:664;

[0337] LCDR1 of SEQ ID NO: 773, 710, 674 or 671;

[0338] LCDR2 of SEQ ID NO: 669 or 673; and

[0339] LCDR3 of SEQ ID NO:670.

[0340] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises the following HCDR1, HCDR2, and HCDR3:

[0341] They are SEQ ID NO: 662, 663 and 664 respectively;

[0342] They are SEQ ID NO: 662, 695 and 664, respectively;

[0343] They are SEQ ID NO: 665, 663 and 664 respectively;

[0344] They are SEQ ID NO: 665, 695 and 664, respectively;

[0345] They are SEQ ID NO: 662, 689 and 664, respectively;

[0346] They are SEQ ID NO: 666, 663 and 664 respectively;

[0347] They are SEQ ID NO: 666, 695 and 664, respectively;

[0348] They are SEQ ID NO: 665, 689, and 664 respectively; or

[0349] They are SEQ ID NO: 666, 689 and 664, respectively.

[0350] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises the following LCDR1, LCDR2 and LCDR3:

[0351] They are SEQ ID NO: 773, 669 and 670, respectively;

[0352] They are SEQ ID NO: 773, 673 and 670 respectively;

[0353] They are SEQ ID NO:710, 673 and 670 respectively;

[0354] They are SEQ ID NO: 674, 673 and 670 respectively;

[0355] They are SEQ ID NO: 671, 673 and 690 respectively;

[0356] They are SEQ ID NO: 773, 673 and 690 respectively;

[0357] They are SEQ ID NO: 671, 669, and 670 respectively; or

[0358] They are SEQ ID NO:776, 673 and 670 respectively.

[0359] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 773, 669 and 670.

[0360] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 773, 673 and 670.

[0361] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 671, 669 and 670.

[0362] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 671, 673 and 690.

[0363] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, as specified in SEQ ID NO: 662, 695, 664, 773, 669 and 670.

[0364] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 695, 664, 773, 673 and 670.

[0365] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 695, 664, 671, 669 and 670.

[0366] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 695, 664, 671, 673 and 670.

[0367] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 665, 663, 664, 773, 669 and 670.

[0368] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 665, 663, 664, 773, 673 and 670.

[0369] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 665, 663, 664, 671, 669 and 670.

[0370] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO: 665, 663, 664, 671, 673 and 670.

[0371] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 665, 695, 664, 773, 669 and 670.

[0372] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO: 665, 695, 664, 773, 673 and 670.

[0373] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 665, 695, 664, 776, 669 and 670.

[0374] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 665, 695, 664, 776, 673 and 670.

[0375] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 666, 663, 664, 773, 669 and 670.

[0376] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 666, 663, 664, 773, 673 and 670.

[0377] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 666, 663, 664, 776, 669 and 670.

[0378] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO: 666, 663, 664, 671, 673 and 670.

[0379] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 666, 695, 664, 773, 669 and 670.

[0380] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO: 666, 695, 664, 773, 673 and 670.

[0381] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 666, 695, 664, 671, 669 and 670.

[0382] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO: 666, 695, 664, 671, 673 and 670.

[0383] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 689, 664, 671, 673 and 670.

[0384] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 710, 673 and 670.

[0385] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 671, 673 and 690.

[0386] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 773, 673 and 690.

[0387] In some embodiments, the anti-CD3 antibody of the present invention or its antigen-binding fragment 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 its antigen-binding fragment of the present invention comprises HC and 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 with 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 with SEQ ID NO: 645, 716, 649, 676, 677 or 650. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises HC and 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 with 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 with SEQ ID NO: 645, 716, 649, 676, 677 or 650, wherein sequence variations do not occur in the CDR region.

[0388] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment 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 its antigen-binding fragment of the present invention comprises VH and VL, wherein the VH has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher such as 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:651, 652, 657, 653, 654, 655, 687 or 656, and the VL has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher such as 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:658, 659, 694, 660, 688, 678 or 661. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises VH and VL, wherein the VH has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher such as 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:651, 652, 657, 653, 654, 655, 687 or 656, and the VL has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher such as 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:658, 659, 694, 660, 688, 678 or 661, wherein sequence variations do not occur in the CDR region.

[0389] PSMA-specific antibodies

[0390] Antibodies and fragments thereof bind to PSMA target antigens in chimpanzees. In one embodiment, the antibodies and fragments bind to 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 a factor of 5. In this case, the same antibody molecule can be used both for preclinical evaluation of the safety, activity, and / or pharmacokinetic characteristics of PSMA in primates and as a drug in humans. In other words, the same PSMA-specific molecule can be used for both preclinical animal studies and human clinical studies. This results in highly comparable results in animal studies and significantly increased predictive power compared to species-specific alternative molecules. Because the PSMA domains are species-specific, i.e., reacting with human and macaque antigens, the antibodies or fragments thereof of the present invention can be used both for preclinical evaluation of the safety, activity, and / or pharmacokinetic characteristics of these binding domains in primates and in the same form as a human drug.

[0391] This invention also provides multispecific antibodies that specifically bind to PSMA. According to the invention, bispecific (i.e., bifunctional) antibodies can be used to bind to 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. Various antibody fragment-based molecules are known and are under investigation, for example, for cancer therapy.

[0392] This invention also provides a PSMAדeffect antigen” bispecific antibody. In one embodiment, the effector antigen of the PSMAדeffect antigen” bispecific antibody is CD3. It has been discovered in this invention that PSMA×CD3 bispecific antibodies can be generated, wherein the same molecule can be used for preclinical animal testing and clinical studies, and even for human treatment. This is due to the identification of a PSMA×CD3 bispecific antibody that, in addition to binding to human PSMA and human CD3, also binds to antigenic homologs of chimpanzees and macaques. The PSMA×CD3 bispecific antibody of this invention can be used as a therapeutic agent against a variety of diseases, including but not limited to cancer. Given the above, there is no longer a need to construct alternative target PSMA×CD3 bispecific antibodies for testing in species phylogenetically distant from (humans). Therefore, the same molecule can be used for preclinical animal testing, as well as for human administration after market approval and therapeutic drug administration.

[0393] In some of the embodiments described herein, the separating antibody or antibody fragment thereof that specifically binds to PSMA has one, two, three, four, or five of the following characteristics:

[0394] a. With an equilibrium dissociation constant (K) less than 25 nM or smaller. D ) binds to the extracellular domain (ECD) of chimpanzee PSMA, in which KD Measurements were taken using the ProteOnXPR36 system at +25°C.

[0395] b. LNCaP lymphocytes were bound with a calculated EC50 of 20 nM or less, and HEK cells expressing PSMA from cynomolgus monkeys were bound with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between bound LNCaP cells and bound HEK cells expressing PSMA from cynomolgus monkeys was less than 5-fold, and wherein the calculated EC50 was measured in a whole-cell binding assay using flow cytometry at 0°C.

[0396] c. With an equilibrium dissociation constant of 12 nM or less (K D ) combines recombinant PSMA ECD from humans (SEQ ID NO:55), recombinant PSMA ECD from chimpanzees (SEQ ID NO:52), or recombinant PSMA ECD from cynomolgus monkeys (SEQ ID NO:53), where K D Resonance determination of the ProteOn XPR36 system using Proteon surface plasmon oscillators at +25°C;

[0397] d. When paired with anti-CD3 antibody in a bispecific antibody, it showed T cell-mediated killing of LNCaP cells, C42 cells, HEK cells expressing human PSMA, or HEK cells expressing cynomolgus monkey PSMA, wherein T cell-mediated killing was measured by a chromium-51 or caspase 3 / 7 activation assay, or

[0398] e. Identify conformational epitopes, wherein the epitopes are composed of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:51).

[0399] Exemplary examples of such antibodies or fragments thereof are the PSMA antibodies described herein: 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.

[0400] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:56, 57, 58, 59, 60, and 61.

[0401] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:62, 63, 64, 65, 60, and 66.

[0402] In some embodiments of the invention described herein, the antibodies of the invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO: 67, 68, 69, 70, 71, and 72.

[0403] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:73, 74, 75, 76, 60, and 61.

[0404] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 79, 80, 81, 82, and 83.

[0405] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:84, 85, 86, 87, 60, and 88.

[0406] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:89, 90, 91, 92, 93, and 94.

[0407] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO: 95, 96, 97, 65, 60, and 66.

[0408] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:84, 98, 99, 100, 82, and 101.

[0409] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:89, 90, 102, 103, 104, and 105.

[0410] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:89, 90, 106, 103, 104, and 105.

[0411] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO: 107, 108, 109, 76, 60, and 88.

[0412] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 1, 80, 81, 82, and 83.

[0413] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 1, 80, 81, 82, and 83.

[0414] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 1, 80, 4, 82, and 686.

[0415] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 1, 80, 81, 792, and 686.

[0416] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 2, 80, 81, 82, and 83.

[0417] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 3, 80, 81, 82, and 5.

[0418] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 3, 80, 81, 82, and 83.

[0419] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 3, 80, 4, 82, and 686.

[0420] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 3, 80, 81, 792, and 686.

[0421] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 2, 81, 81, 82, and 5.

[0422] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 2, 80, 4, 792, and 686.

[0423] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 2, 80, 4, 792, and 686.

[0424] In some embodiments of the present invention described herein, the antibodies of the present invention that specifically bind to PSMA comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO:78, 683, 80, 81, 792, and 686.

[0425] In some embodiments of the invention described herein, the PSMA-specific antibodies of the invention comprise the 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 PSMA-specific antibodies of the invention comprise the light chain variable region (VL) of SEQ ID NO: 9, 111, 113, 115, 117, 119, 122, 124, 127, 129, 131, or 682.

[0426] In some embodiments of the invention described herein, the PSMA-specific antibodies of the invention comprise the heavy chain sequence of SEQ ID NO: 12, 13, 132, 134, 136, 138, 140, 141, 143, 145, 146, 148, 150, 151 or 679.

[0427] In some embodiments of the invention described herein, the PSMA-specific antibodies of the invention comprise a light chain sequence of SEQ ID NO: 14, 15, 75, 133, 135, 137, 139, 142, 144, 147, 149 or 680.

[0428] CD33-specific antibody

[0429] The CD33-specific antibody of the present invention has one or more desired functional properties, including but not limited to high affinity binding to CD33 and / or CD3, high specificity to CD33 and / or CD3, and the ability to treat or prevent cancer when administered alone or in combination with other anticancer therapies.

[0430] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment binds to the C2 domain of CD33. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment binds to the V domain of CD33. Full-length human CD33 is provided by UniprotP20138 (SEQ ID NO:244).

[0431] As used in this article, an antibody that "specifically binds to CD33" refers to an antibody with a concentration of 1×10⁻⁶. -7 M or smaller, preferably 1×10 -8 M or smaller, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 An antibody that binds to CD33, preferably to human CD33, and preferably to the C2 domain of CD33, with an M or smaller KD.

[0432] The antibodies or antigen-binding fragments described herein can exist in various forms, but will include one or more of the antibody CDRs shown in Tables 39 and 40.

[0433] This document describes recombinant antibodies and antigen-binding fragments that specifically bind to CD33. In some embodiments, the CD33-specific antibody or antigen-binding fragment is human IgG or a derivative thereof. Although the CD33-specific antibody or antigen-binding fragment illustrated herein is human, the illustrated antibody or antigen-binding fragment may also be chimeric.

[0434] In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided, comprising heavy chains of 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, comprising heavy chains of CDR1, CDR2, and CDR3 of any of the antibodies described in Table 39 and light chains of CDR1, CDR2, and CDR3 of any of the antibodies described in Table 40.

[0435] In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided, which include the heavy chain variable regions shown in Table 38. In some embodiments, CD33-specific antibodies or antigen-binding fragments thereof are provided, which include the light chain variable regions shown in Table 38.

[0436] The heavy chain variable domains and light chain variable domains 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 the CD33 bispecific antibody, the effector arm is the CD3 arm. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 671, 673, and 670. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises VH and VL, respectively, of SEQ ID NO: 652 and 661. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises HC and LC, respectively, of SEQ ID NO: 640 and 676.

[0437] In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, as specified in SEQ ID NOs: 662, 663, 664, 773, 673, and 670. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises VH and VL, respectively, as specified in SEQ ID NOs: 657 and 678. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises HC and LC, respectively, as specified in SEQ ID NOs: 675 and 677.

[0438] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises a heavy chain variable region having at least 95% identity with a polypeptide sequence of 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 at least 95% identity with a polypeptide sequence of 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. Furthermore, the anti-CD3 antibody or its antigen-binding fragment contains a heavy chain variable region having a polypeptide sequence having at least 95% identity with SEQ ID NO:257 or 258, or a light chain variable region having a polypeptide sequence having at least 95% identity with SEQ ID NO:298 or 299.

[0439] IL1RAP specific antibody

[0440] As used herein, the terms “interleukin-1 receptor helper 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 is also referred to in the scientific literature as IL1R3, C3orf13, FLJ37788, IL-1RAcP, and EG3556.

[0441] The antibodies or antigen-binding fragments described herein can exist in various forms, but will include one or more of the antibody CDRs shown in Table 29.

[0442] This document describes recombinant antibodies and antigen-binding fragments that specifically bind to IL1RAP. In some embodiments, the IL1RAP-specific antibody or antigen-binding fragment is human IgG or a derivative thereof. Although the IL1RAP-specific antibody or antigen-binding fragment illustrated herein is human, the illustrated antibody or antigen-binding fragment may also be chimeric.

[0443] In some embodiments, IL1RAP-specific antibodies or antigen-binding fragments thereof are provided, comprising heavy chains containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 29. In some embodiments, IL1RAP-specific antibodies or antigen-binding fragments thereof are provided, comprising heavy chains containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 29, and light chains containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 29.

[0444] In some embodiments, IL1RAP-specific antibodies or antigen-binding fragments thereof are provided, comprising a heavy chain variable region of any of the antibodies shown in Table 30. In some embodiments, IL1RAP-specific antibodies or antigen-binding fragments thereof are provided, comprising a light chain variable region of any of the antibodies shown in Table 30. In some embodiments, IL1RAP-specific antibodies or antigen-binding fragments thereof are provided, comprising both a heavy chain variable region and a light chain variable region of any of the antibodies shown in Table 30.

[0445] The heavy chain variable domains and light chain variable domains of the antibodies discussed in this section and shown in Table 30 are suitable for inclusion in bispecific constructs, wherein the target arm is an anti-IL1RAP arm. For example, in some embodiments of the IL1RAP bispecific antibody, the effector arm is a CD3 arm. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of SEQ ID NO: 662, 663, 664, 671, 673, and 670. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises VH and VL, respectively, of SEQ ID NO: 652 and 661. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HC and LC, respectively, of SEQ ID NO: 640 and 676.

[0446] In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, as specified in SEQ ID NOs: 662, 663, 664, 773, 673, and 670. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises VH and VL, respectively, as specified in SEQ ID NOs: 657 and 678. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises HC and LC, respectively, as specified in SEQ ID NOs: 675 and 677.

[0447] TMEFF2 specific antibody

[0448] This invention provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, which binds to the proximal membrane region of TMEFF2 (SEQ ID NO: 629). The anti-TMEFF2 antibody of this invention, binding to the proximal membrane region of TMEFF2, is not internalized by cells. While not wishing to be bound by any particular theory, it is anticipated that non-internalized anti-TMEFF2 antibodies, compared to internalized anti-TMEFF2 antibodies, possess improved oncogenic effects mediated by antibody effector function due to the lack of internalization and degradation of TMEFF2.

[0449] "Binding to the proximal membrane region" means that 90% of the antibody epitope residues identified using hydrogen / deuterium exchange (H / D exchange) are located within the proximal membrane region of TMEFF2. Epitope residues are those protected by the test antibody that have at least a 5% difference in deuteration level through H / D exchange. Exemplary examples of such antibodies are TMEB675, TMEB570, TMEB674, TMEB565, TMEB762, and TMEB757 as described herein.

[0450] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment binds to the proximal membrane region of TMEFF2 within residues HGKCEHSINMQEPSC (SEQ ID NO: 592) or DAGYTGQHCEKKDYSVL (SEQ ID NO: 600). An exemplary anti-TMEFF2 antibody that binds within residue HGKCEHSINMQEPSC (SEQ ID NO: 592) is TMEB570. An exemplary anti-TMEFF2 antibody that binds within residue DAGYTGQHCEKKDYSVL (SEQ ID NO: 600) is TMEB675. TNEB675 variants TMEB762 and TMEB757 are also expected to bind to the proximal membrane region of TMEFF2 within residue DAGYTGQHCEKKDYSVL (SEQ ID NO: 600).

[0451] In the H / D exchange assay, TMEFF2ECD expressed via recombinantly is incubated in deuterated water for a predetermined time in the presence or absence of antibodies, resulting in the introduction of deuterium at exchangeable hydrogen atoms unprotected by antibodies. The protein is then digested with a protease, and the peptide fragments are analyzed by LC-MS. The H / D exchange assay can be performed using known protocols. Exemplary protocols are described in Example 5.

[0452] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment competitively binds to the proximal membrane region of TMEFF2 with a 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.

[0453] The competitive binding of the test antibody to the proximal membrane region of TMEFF2 against the reference antibody can be measured in vitro using well-known methods. For example, the MSD Sulfo-Tag can be assessed by ELISA in the presence of an unlabeled reference antibody. TM The binding of the NHS-ester-labeled test antibody to the proximal region of the TMEFF2 membrane can be demonstrated using Bioacore analysis or flow cytometry. The test antibody competes with the reference antibody for binding to TMEFF2 when it inhibits the binding of the reference antibody to the proximal region of the TMEFF2 membrane by 85% or greater, such as 90% or greater, or 95% or greater.

[0454] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising the following heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3:

[0455] They are SEQ ID NO: 582, 584, 587, 18, 588 and 22 respectively;

[0456] They are SEQ ID NO: 583, 585, 16, 19, 21 and 23 respectively;

[0457] They are SEQ ID NO: 582, 586, 17, 18, 588 and 24 respectively;

[0458] They are SEQ ID NO: 583, 585, 16, 18, 588 and 22 respectively; or

[0459] They are SEQ ID NO: 582, 584, 587, 18, 588 and 603, respectively.

[0460] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment of the present invention is expressed at approximately 0.4 × 10⁻⁶. -9 M or a smaller equilibrium dissociation constant (K) D ) binds to the proximal region of the membrane of TMEFF2, where K D Measurements were performed at room temperature in acetate buffer solution at pH 4.5–5.0 using surface plasmon resonance.

[0461] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment is at a concentration between about 0.1 × 10⁻⁶. - 10 M and approximately 0.4 × 10 -9 K between M D It binds to the proximal region of the membrane of TMEFF2.

[0462] The affinity of the antibody for the proximal membrane region of TMEFF2 can be determined experimentally using any suitable method. An exemplary method employs a ProteOnXPR36, Biacore3000, or KinExA instrument, ELISA, or competitive binding assay known to those skilled in the art. The measured antibody affinity for TMEFF2 can vary if measured under different conditions (e.g., isotonicity, pH). Therefore, affinity and other binding parameters (e.g., K0) will affect the determination of TMEFF2 affinity. D K on and K off Measurements of affinity are typically performed using standardized conditions and standardized buffers (such as those described herein). Those skilled in the art will understand that the internal error (measured as standard deviation, SD) of affinity measurements using, for example, Biacore 3000 or ProteOn, is typically within 5%–33% of the measured value within the typical detection limit. Therefore, when referring to K… D When referring to values, the term "approximately" reflects the typical standard deviation in the measurement. For example, 1 × 10⁻⁶. -9 M of K D The typical SD is at most ±0.33×10 -9 M.

[0463] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the proximal membrane region of TMEFF2 and includes a heavy chain variable region (VH) framework derived from VH3_3-23 (SEQ ID NO:53) or VH1_1-69 (SEQ ID NO:54).

[0464] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the proximal membrane region of TMEFF2 and includes a light chain variable region (VL) framework derived from VKI_L11 (SEQ ID NO:55) or VKIIII_A27 (SEQ ID NO:591).

[0465] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, which binds to the proximal membrane region of TMEFF2 and includes a VH frame and a VL frame derived from VH3_3-23 of SEQ ID NO:53 and VKI_L11 of SEQ ID NO:55, respectively.

[0466] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, which binds to the proximal membrane region of TMEFF2 and includes a VH frame and a VL frame derived from VH1_1-69 of SEQ ID NO:54 and VKIII_A27 of SEQ ID NO:591, respectively.

[0467] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, which binds to the proximal membrane region of TMEFF2 and includes a VH frame and a VL frame derived from VH1_1-69 of SEQ ID NO:54 and VKI_L11 of SEQ ID NO:55, respectively.

[0468] Antibodies containing heavy or light chain variable regions “derived” from a specific frame or germline sequence are antibodies obtained from systems using human germline immunoglobulin genes, such as those from transgenic mice, rats, or chickens, or phage display libraries as discussed herein. Antibodies containing a specific frame derived from a germline sequence may contain amino acid differences compared to the sequence from which it originated, due to, for example, naturally occurring somatic mutations or intentional substitutions.

[0469] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively, SEQ ID NO:582,584,587,18,588 and22.

[0470] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28.

[0471] In some implementations, VH is encoded by the polynucleotide of SEQ ID NO:39, and VL is encoded by the polynucleotide of SEQ ID NO:42.

[0472] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises HC of SEQ ID NO:32 and LC of SEQ ID NO:35.

[0473] In some implementations, HC is encoded by a polynucleotide of SEQ ID NO:46, and VL is encoded by a polynucleotide of SEQ ID NO:49.

[0474] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO:583,585,16,19,21 and23 respectively.

[0475] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises VH of SEQ ID NO:589 and VL of SEQ ID NO:29.

[0476] In some implementations, VH is encoded by the polynucleotide of SEQ ID NO:40, and VL is encoded by the polynucleotide of SEQ ID NO:43.

[0477] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises HC of SEQ ID NO:33 and LC of SEQ ID NO:36.

[0478] In some implementations, HC is encoded by the polynucleotide of SEQ ID NO:47, and VL is encoded by the polynucleotide of SEQ ID NO:50.

[0479] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO:582,586,17,18,588 and24 respectively.

[0480] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30.

[0481] In some implementations, VH is encoded by the polynucleotide of SEQ ID NO:41, and VL is encoded by the polynucleotide of SEQ ID NO:44.

[0482] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises HC of SEQ ID NO:34 and LC of SEQ ID NO:37.

[0483] In some implementations, HC is encoded by the polynucleotide of SEQ ID NO:48, and LC is encoded by the polynucleotide of SEQ ID NO:51.

[0484] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO:583,585,16,18,588 and22 respectively.

[0485] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises VH of SEQ ID NO:589 and VL of SEQ ID NO:31.

[0486] In some implementations, VH is encoded by the polynucleotide of SEQ ID NO:40, and VL is encoded by the polynucleotide of SEQ ID NO:45.

[0487] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises HC of SEQ ID NO:33 and LC of SEQ ID NO:38.

[0488] In some implementations, HC is encoded by the polynucleotide of SEQ ID NO:47, and LC is encoded by the polynucleotide of SEQ ID NO:590.

[0489] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively, SEQ ID NO:582,584,587,18,588 and603.

[0490] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises VH of SEQ ID NO:604 and VL of SEQ ID NO:607.

[0491] In some implementations, VH is encoded by the polynucleotide of SEQ ID NO:618, and VL is encoded by the polynucleotide of SEQ ID NO:619.

[0492] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises HC of SEQ ID NO:614 and LC of SEQ ID NO:615.

[0493] In some implementations, HC is encoded by the polynucleotide of SEQ ID NO:620, and LC is encoded by the polynucleotide of SEQ ID NO:621.

[0494] The present invention also provides an isolated anti-TMEFF2 antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively, SEQ ID NO:582,584,587,18,588 and603.

[0495] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises VH of SEQ ID NO:612 and VL of SEQ ID NO:613.

[0496] In some implementations, VH is encoded by the polynucleotide of SEQ ID NO:622, and VL is encoded by the polynucleotide of SEQ ID NO:623.

[0497] In some embodiments, the isolated anti-TMEFF2 antibody or its antigen-binding fragment comprises HC of SEQ ID NO:616 and LC of SEQ ID NO:617.

[0498] In some implementations, HC is encoded by the polynucleotide of SEQ ID NO:624, and LC is encoded by the polynucleotide of SEQ ID NO:625.

[0499] In some implementations, the isolated anti-TMEFF2 antibody or its antigen-binding fragment is a multispecific antibody.

[0500] In some implementations, the isolated anti-TMEFF2 antibody or its antigen-binding fragment is a bispecific antibody.

[0501] In some implementations, the isolated anti-TMEFF2 bispecific antibody or its antigen-binding fragment binds to T-cell antigens.

[0502] In some implementations, the isolated anti-TMEFF2 bispecific antibody or its antigen-binding fragment binds to CD3.

[0503] In some implementations, the isolated anti-TMEFF2 bispecific antibody or its antigen-binding fragment binds to CD3ε.

[0504] 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.

[0505] Although the embodiments shown in the examples include paired variable domains, one from the heavy chain and one from the 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 for variable domains capable of forming bidomain-specific antigen-binding fragments that can bind to TMEFF2. Screening can be performed using phage display methods, employing, for example, the graded dual-combination method disclosed in International Patent Publication WO1992 / 01047. In this method, a complete library of clones encoding the other chain (VL or VH) is infected using a single colony containing a VH or VL chain clone, and the resulting bidomain-specific antigen-binding domain is selected according to phage display technology using known methods and those described herein. Therefore, using the method disclosed in International Patent Publication WO1992 / 01047, individual VH and VL polypeptide chains can be used to identify additional anti-TMEFF2 antibodies.

[0506] Bispecific anti-TMEFF2 / anti-CD3 antibody

[0507] This invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment comprising a first domain for binding TMEFF2 and a second domain for binding CD3, wherein the antibody binds to the proximal membrane region of TMEFF2. While not wishing to be bound by any specific theory, bispecific antibodies binding to the proximal membrane region of TMEFF2 can more effectively mediate T cell-mediated tumor cell killing.

[0508] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a first domain for binding TMEFF2 and a second domain for binding CD3, wherein the antibody competes with a reference antibody for binding to the proximal membrane 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.

[0509] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment is at a concentration of approximately 0.4 × 10⁻⁶. -9 M or a smaller dissociation constant (K) D ) binds to the proximal region of the membrane of TMEFF2, where K D Measurements were performed at room temperature in acetate buffer solution at pH 4.5–5.0 using surface plasmon resonance.

[0510] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment is at a concentration between about 0.1 × 10⁻⁶. -10 M and approximately 0.4 × 10 -9 K between M D The proximal region of the membrane that binds to TMEFF2.

[0511] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment thereof, wherein the first domain comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3:

[0512] They are SEQ ID NO: 582, 584, 587, 18, 588 and 22 respectively;

[0513] They are SEQ ID NO: 583, 585, 16, 19, 21 and 23 respectively;

[0514] They are SEQ ID NO: 582, 586, 17, 18, 588 and 24 respectively;

[0515] They are SEQ ID NO: 583, 585, 16, 18, 588 and 22 respectively; or

[0516] They are SEQ ID NO: 582, 584, 587, 18, 588 and 603, respectively.

[0517] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment thereof, wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO:662,663,664,671,673 and690, respectively.

[0518] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the first domain comprises

[0519] VH of SEQ ID NO:25 and VL of SEQ ID NO:28;

[0520] VH of SEQ ID NO:589 and VL of SEQ ID NO:29;

[0521] VH of SEQ ID NO:27 and VL of SEQ ID NO:30;

[0522] VH of SEQ ID NO:589 and VL of SEQ ID NO:31;

[0523] VH of SEQ ID NO:604 and VL of SEQ ID NO:607; or

[0524] VH of SEQ ID NO:612 and VL of SEQ ID NO:613.

[0525] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the second domain comprises

[0526] VH of SEQ ID NO:652 and VL of SEQ ID NO:661.

[0527] In some implementations, the second structural domain includes VH of SEQ ID NO:657 and VL of SEQ ID NO:658.

[0528] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a first domain for binding TMEFF2 and a second domain for binding CD3, wherein

[0529] The first structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:582, 584, 587, 18, 588, and 22; and the second structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:662, 663, 664, 671, 673, and 690.

[0530] The first structural domain contains VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second structural domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0531] The first structural domain contains VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second structural domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678;

[0532] Bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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

[0533] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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.

[0534] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a first domain for binding TMEFF2 and a second domain for binding CD3, wherein

[0535] The first structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:583, 585, 16, 19, 21, and 23; and the second structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:662, 663, 664, 671, 673, and 690.

[0536] The first structural domain contains VH of SEQ ID NO:589 and VL of SEQ ID NO:29, and the second structural domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0537] The first structural domain contains VH of SEQ ID NO:589 and VL of SEQ ID NO:29, and the second structural domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0538] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0539] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0540] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a first domain for binding TMEFF2 and a second domain for binding CD3, wherein

[0541] The first structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:582, 586, 17, 18, 588, and 24; and the second structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:662, 663, 664, 671, 673, and 690.

[0542] The first structural domain contains VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second structural domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0543] The first structural domain contains VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second structural domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0544] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0545] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0546] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a first domain for binding TMEFF2 and a second domain for binding CD3, wherein

[0547] The first structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:583, 585, 16, 18, 588, and 22; and the second structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, SEQ ID NO:662, 663, 664, 671, 673, and 690.

[0548] The first structural domain contains VH of SEQ ID NO:589 and VL of SEQ ID NO:31, and the second structural domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0549] The first structural domain contains VH of SEQ ID NO:589 and VL of SEQ ID NO:31, and the second structural domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0550] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises 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;

[0551] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises 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.

[0552] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a first domain for binding TMEFF2 and a second domain for binding CD3, wherein

[0553] The first structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, according to SEQ ID NO:582, 584, 587, 18, 588, and 603; and the second structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, according to SEQ ID NO:662, 663, 664, 671, 673, and 690.

[0554] The first structural domain contains VH of SEQ ID NO:604 and VL of SEQ ID NO:607, and the second structural domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0555] The first structural domain contains VH of SEQ ID NO:604 and VL of SEQ ID NO:607, and the second structural domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0556] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0557] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0558] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a first domain for binding TMEFF2 and a second domain for binding CD3, wherein

[0559] The first structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, according to SEQ ID NO:582, 584, 587, 18, 588, and 603; and the second structural domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively, according to SEQ ID NO:662, 663, 664, 671, 673, and 690.

[0560] The first structural domain contains VH of SEQ ID NO:612 and VL of SEQ ID NO:613, and the second structural domain contains VH of SEQ ID NO:652 and VL of SEQ ID NO:661;

[0561] The first structural domain contains VH of SEQ ID NO:612 and VL of SEQ ID NO:613, and the second structural domain contains VH of SEQ ID NO:657 and VL of SEQ ID NO:678; and / or

[0562] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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;

[0563] The bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment 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.

[0564] Implementation Plan :

[0565] The present invention provides the following non-limiting embodiments.

[0566] 1. An isolated recombinant anti-CD3 antibody or its antigen-binding fragment, comprising:

[0567] a) A heavy chain and a light chain, the heavy chain comprising: a heavy chain complementarity determination region (HCDR) 1 comprising SEQ ID NO:662, an HCDR 2 comprising SEQ ID NO:663, and an HCDR 3 comprising SEQ ID NO:664, the light chain comprising: a light chain complementarity determination region (LCDR) 1 comprising SEQ ID NO:671, an LCDR 2 comprising SEQ ID NO:673, and an LCDR 3 comprising SEQ ID NO:690;

[0568] b) The heavy chain variable region containing SEQ ID NO:652 and the light chain variable region containing SEQ ID NO:661;

[0569] c) The heavy chain containing SEQ ID NO:640 and the light chain containing SEQ ID NO:676;

[0570] d) The heavy chain and the light chain, wherein the heavy chain comprises: HCDR1 comprising SEQ ID NO:662, HCDR2 comprising SEQ ID NO:663 and HCDR3 comprising SEQ ID NO:664, and the light chain comprises: LCDR1 comprising SEQ ID NO:773, LCDR2 comprising SEQ ID NO:673 and LCDR3 comprising SEQ ID NO:690;

[0571] e) Containing the heavy chain variable region of SEQ ID NO:657 and the light chain variable region of SEQ ID NO:678; or

[0572] f) The heavy chain containing SEQ ID NO:675 and the light chain containing SEQ ID NO:678.

[0573] 2. An isolated recombinant anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to cynomolgus monkey or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of about 300 nM or less.

[0574] 3. The isolated recombinant anti-CD3 antibody or its antigen-binding fragment according to embodiment 2, wherein the binding affinity is about 100 nM or less.

[0575] 4. The isolated recombinant anti-CD3 antibody or its antigen-binding fragment according to embodiment 2 or 3, wherein the binding affinity is measured by flow cytometry or by Proteon surface plasmon resonance assay at +25°C using the ProteOnXPR36 system.

[0576] 5. The isolated recombinant anti-CD3 antibody or its antigen-binding fragment 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:

[0577] a) Binding of human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 300 nM or less, and binding of cynomolgus monkey CD3-expressing HEK cells with a calculated EC50 of 300 nM or less, wherein the difference in calculated EC50 between binding CD3+ T lymphocytes and binding of cynomolgus monkey CD3-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole-cell binding assay using flow cytometry at 0°C;

[0578] b) With an equilibrium dissociation constant (K) of 300 nM or less. D ) combined with recombinant CD3d from human (SEQ ID NO: 691), or combined with recombinant CD3e from human (SEQ ID NO: 636), or combined with recombinant CD3d from cynomolgus monkey (SEQ ID NO: 692), or combined with recombinant CD3e from cynomolgus monkey (SEQ ID NO: 693), wherein K D Resonance determination of the ProteOn XPR36 system using Proteon surface plasmon oscillators at +25°C;

[0579] c) Binding to CD3e residues 1-6, as determined by X-ray crystallography; or

[0580] d) Activate T cells or induce CD69 expression to a level similar to cOKT3 or SP34-2, as determined by fluorescence-activated cell sorting assay.

[0581] 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:

[0582] a) Heavy chain replacement with K409R, F405L or F405L and R409K;

[0583] b) Heavy chain substitution of S228P, F234A and L235A;

[0584] c) Heavy chain substitution of L234A, G237A, P238S, H268A, A330S, and P331S, wherein the antibody is an IgG1 isotype; or

[0585] d) Heavy chain replacement of S228P, wherein the antibody is an IgG4 isotype;

[0586] The residues are numbered according to the EU index.

[0587] 7. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673 and 690 respectively.

[0588] 8. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments comprises a heavy chain variable region (VH) and a light chain variable region (VL) of SEQ ID NO: 652 and 661, respectively.

[0589] 9. The antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments comprises a heavy chain sequence (HC) and a light chain sequence (LC) of SEQ ID NO: 640 and 676, respectively.

[0590] 10. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively, SEQ ID NO: 662, 663, 664, 773, 673 and 690.

[0591] 11. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising VH and VL of SEQ ID NO: 657 and 678, respectively.

[0592] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising HC and LC of SEQ ID NO: 675 and 677, respectively.

[0593] 13. An antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO:662,663,664,671,673 and690.

[0594] 14. An antibody or an antigen-binding fragment thereof, comprising VH and VL, respectively, SEQ ID NO:652 and 661.

[0595] 15. An antibody or an antigen-binding fragment thereof, comprising HC and LC, respectively, SEQ ID NO:640 and 676.

[0596] 16. An antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, SEQ ID NO:662,663,664,773,673 and690.

[0597] 17. An antibody or an antigen-binding fragment thereof comprising VH and VL, respectively, SEQ ID NO:657 and 678.

[0598] 18. An antibody or an antigen-binding fragment thereof, comprising HC and LC, respectively, SEQ ID NO:675 and 677.

[0599] 19. An antibody or an antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the antibody is human or humanized.

[0600] 20. The antibody according to embodiment 19, wherein the antibody is an IgG4 or IgG1 isotype.

[0601] 21. The antibody according to embodiment 20, comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions of antibody Fc.

[0602] 22. The antibody according to embodiment 18, comprising:

[0603] a) Replacement of light chains D43G, L49M, L50I, S62N, and Q85E;

[0604] b) Replacement of light chains D43G, V48L, L49M, L50I, S62N, Q85E, and H89Y;

[0605] c) Heavy chain substitution of R10G, R13K, V73I, R70K, T83S, and L96V;

[0606] d) Replace the light chain with any one of D43G, V48L, L49M, L50I, S62N, Q85E, or H89Y; or

[0607] e) Heavy chain substitution with any one of R10G, R13K, V73I, R79K, T83S, or L96V.

[0608] The residues substituted with light chains are numbered according to SEQ ID No:661, and the residues substituted with heavy chains are numbered according to SEQ ID No:652.

[0609] 23. The antibody according to any one of the foregoing embodiments, wherein the antibody is bispecific or multispecific.

[0610] 24. A bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO:662,663,664,671,673 and690, respectively.

[0611] 25. The bispecific antibody according to embodiment 24, wherein the first domain and the second domain are IgG4 isotypes, and wherein the first domain or the second domain comprises S228P, F234A, L235A, F405L and R409K heavy chain substitutions, and another domain of the first domain or the second domain comprises S228P, F234A and L235A heavy chain substitutions, wherein the residues are numbered according to the EU index.

[0612] 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 F405L or F405L and R409K substitution, wherein the residues are numbered according to the EU index.

[0613] 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.

[0614] 28. The bispecific antibody according to embodiment 24, wherein the first domain and the second domain are IgG4 isotypes, wherein one of the first domain or the second domain contains an S228P heavy chain substitution, and the other of the first domain or the second domain contains an S228P, F405L and R409K heavy chain substitution, wherein the residues are numbered according to the EU index.

[0615] 29. The bispecific antibody according to claim 24, wherein the first domain comprises VH and VL, respectively, of SEQ ID NO: 652 and 661.

[0616] 30. The bispecific antibody according to claim 24, wherein the first domain comprises HC and LC of SEQ ID NO: 640 and 676, respectively.

[0617] 31. The bispecific antibody according to claim 24, wherein the first domain comprises VH and VL, respectively, of SEQ ID NO: 657 and 678.

[0618] 32. The bispecific antibody according to claim 24, wherein the first domain comprises HC and LC of SEQ ID NO: 675 and 677, respectively.

[0619] 33. The bispecific antibody of claim 24, wherein the second antigen is a cell surface antigen expressed on target cells other than immune effector cells.

[0620] 34. The bispecific antibody according to claim 33, wherein the cell surface antigen is a tumor-associated antigen.

[0621] 35. The bispecific antibody according to any one of claims 24 to 34, wherein the second antigen is CD33, IL1RAP, PSMA, or TMEFF2.

[0622] 36. The bispecific antibody according to embodiment 35, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 78, 683, 80, 81, 792, and 686, respectively.

[0623] 37. The bispecific antibody according to embodiment 33, wherein the first domain comprises VH and VL of SEQ ID NO:652 and 661, respectively; and wherein the second domain comprises VH and VL of SEQ ID NO:681 and 682, respectively.

[0624] 38. The bispecific antibody according to embodiment 33, wherein the first domain comprises HC and LC of SEQ ID NO:640 and 676, respectively; and wherein the second domain comprises HC and LC of SEQ ID NO:679 and 680, respectively.

[0625] 39. The bispecific antibody according to embodiment 33, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 78, 3, 80, 81, 792, and 686, respectively.

[0626] 40. The bispecific antibody according to embodiment 33, wherein the first domain comprises VH and VL of SEQ ID NO:652 and 661, respectively; and wherein the second domain comprises VH and VL of SEQ ID NO:8 and 682, respectively.

[0627] 41. The bispecific antibody according to embodiment 33, wherein the first domain comprises HC and LC of SEQ ID NO: 640 and 676, respectively; and wherein the second domain comprises HC and LC of SEQ ID NO: 13 and 680, respectively.

[0628] 42. The bispecific antibody according to embodiment 33, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 78, 79, 80, 81, 82, and 83, respectively.

[0629] 43. The bispecific antibody according to embodiment 33, wherein the first domain comprises VH and VL of SEQ ID NO: 652 and 661, respectively; and wherein the second domain comprises VH and VL of SEQ ID NO: 116 and 117, respectively.

[0630] 44. The bispecific antibody according to embodiment 33, wherein the first domain comprises HC and LC of SEQ ID NO: 640 and 676, respectively; and wherein the second domain comprises HC and LC of SEQ ID NO: 138 and 139, respectively.

[0631] 45. The bispecific antibody according to embodiment 31, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673, and 670, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 164, 165, 166, 167, 168, and 169, respectively.

[0632] 46. ​​The bispecific antibody according to embodiment 31, wherein the first domain comprises VH and VL of SEQ ID NO:652 and 661, respectively; and wherein the second domain comprises VH and VL of SEQ ID NO:215 and 216, respectively.

[0633] 47. The bispecific antibody according to embodiment 31, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673, and 670, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 349, 390, 341, 471, 513, and 555, respectively.

[0634] 48. The bispecific antibody according to embodiment 31, wherein the first domain comprises VH and VL of SEQ ID NO:652 and 661, respectively; and wherein the second domain comprises VH and VL of SEQ ID NO:267 and 306, respectively.

[0635] 49. The bispecific antibody according to embodiment 31, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 662, 663, 664, 671, 673, and 670, respectively; and wherein the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 363, 404, 445, 485, 527, and 569, respectively.

[0636] 50. The bispecific antibody according to embodiment 31, wherein the first domain comprises VH and VL of SEQ ID NO:652 and 661, respectively; and wherein the second domain comprises VH and VL of SEQ ID NO:281 and 320, respectively.

[0637] 51. A pharmaceutical composition comprising an antibody according to any one of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0638] 52. A polynucleotide encoding an antibody according to any one of embodiments 1 to 50.

[0639] 53. A carrier comprising the polynucleotide according to embodiment 52.

[0640] 54. A host cell comprising the vector according to embodiment 53.

[0641] 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 the antibody to be expressed, and recovering the antibody produced by the host cell.

[0642] 56. A method of treating a subject's cancer, comprising administering to the subject in need 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.

[0643] 57. The method according to embodiment 56, wherein the cancer is a solid tumor or a hematologic malignancy.

[0644] 58. The method according to embodiment 57, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell renal cell carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascularization disease, clear cell renal cell carcinoma (CCRCC), pancreatic cancer, kidney cancer, urethral epithelial carcinoma, or metastatic adenocarcinoma of the liver.

[0645] 59. The method according to embodiment 58, wherein the prostate cancer is refractory prostate cancer, prostate intraepithelial neoplasia, androgen-independent prostate cancer, or malignant prostate cancer.

[0646] 60. The method according to embodiment 57, wherein the hematologic 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 tumor (DPDCN).

[0647] 61. The method according to any one of embodiments 56 to 60, wherein the antibody is administered in combination with a second therapeutic agent.

[0648] 62. The antibody according to any one of embodiments 1 to 50, said antibody being used in treatment.

[0649] 63. An anti-idiotype antibody, said anti-idiotype antibody binding to an antibody according to any one of embodiments 1 to 50.

[0650] Example

[0651] De novo generation and functional characterization of anti-CD3 mAb

[0652] 1-1 Immunization with CD3 antigen To produce CD3 monoclonal antibodies

[0653] Immunization with proprietary vectors encoding the following (Aldevron, Fargo, North Dakota, USA) Human CD3e and human CD3d; cynomolgus monkey CD3e and cynomolgus monkey CD3d. Animals received alternating booster immunizations with human and cynomolgus monkey 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 BLW fusion were placed in three 96-well plates. With IgM not depleted, 133 million cells from the BLX fusion were placed in nine 96-well plates.

[0654] Cell-based ELISA (CELISA) was performed on cells transiently transfected with human and cynomolgus cDNA cloned into selection vectors. The supernatant from hybridomas fusions of BLW (lymphocyte bead depletion) and BLX (lymphocyte bead non-depletion) was analyzed. The selection vectors were: 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 ethyl bromide secondary antibody. For the CELISA transfection control, mammalian cells transfected with the above construct were detected using an anti-labeled antibody.

[0655] Hybridoma supernatants were further analyzed by flow cytometry (FACS) of CD3-positive and CD3-negative Jurkat cells: JurkatCD3+ (E6-1) and JurkatCD3- (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 IgHRP and Bethyl anti-rat IgG1, 2a, 2b, 2c-HRP secondary antibodies.

[0656] The specificity of antibodies in the hybridoma supernatant from hybridomas fusions of BLW and BLX for the human and cynomolgus CD3e / d complexes presented on transiently transfected cells (or, for the human CD3e / d complex presented on JurkatCD3+(E6-1) cells) was confirmed by CELISA of transiently transfected cells and by FACS testing on Jurkat cell lines (Table 3). No significant signal was detected in any of the experimental samples used as negative controls.

[0657] Table 3: Testing of hybridoma supernatants by cell-based ELISA (top) and flow cytometry (bottom) The specificity of the solution. CELISA values ​​represent the relative fluorescence units (rfu) for each sample. FACS values ​​represent the relative fluorescence units (rfu) for each sample. Geometric mean of light intensity .

[0658]

[0659]

[0660] Table 4. CD3 sequences used for immunization

[0661] Human CD3d NP_000723.1(www.uniprot.org / uniprot / P04234)(SEQ ID NO:691) human CD3e (NP_000724.1(www.uniprot.org / uniprot / P07766)(SEQ ID NO:636) Crab-eating monkey CD3d XP_001097302(www.uniprot.org / uniprot / Q95LI8)(SEQ ID NO:692) Crab-eating macaque CD3e CD3e+TCE(www.uniprot.org / uniprot / Q95LI5)(SEQ ID NO:693)

[0662] 1-2 clone anti-CD3 antibody

[0663] Anti-human CD3 antibodies were generated in OmniRats (OMT, Palo Alto, California, USA). Variable region (“V region”) sequences of these clones were extracted from the genomic sequence and analyzed. All obtained sequences were either human IgG heavy chain or λ light chain, and these sequences, especially LC, showed high homology. Sequence alignment with germline pairs revealed 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 the seven mAbs (Table 5A).

[0664] CD3B312 was selected as the most representative clone, and the heavy chain sequence was cloned into human IgG1σ and IgG4PAA with S228P, F234A, L235A mutations and designated protein identifiers, as shown in Table 5B. These were used to generate bispecific antibodies, and T cell redirection function was demonstrated by cytotoxicity.

[0665] Table 5A. Peptide IDs and Protein IDs of Clones

[0666] Cloned ID HC peptide ID 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

[0667] Table 5B. CD3B312 was selected as the most representative clone, and the heavy chain sequence was cloned into a clone with mutations and pointers. In human IgG1σ and IgG4PAA protein identifiers .

[0668] IgG1 IgG1σ IgG4PAA CD3B312 CD3B337 CD3B373

[0669] Table 6. Heavy chain and light chain sequences of 7 monoclonal CD3 antibodies

[0670]

[0671]

[0672]

[0673]

[0674]

[0675] Table 7A. VH and VL sequences of HC and LC isotypes from 7 of the 9 monoclonal CD3 antibodies in the first batch mentioned above. (See Table 3). All HC isotypes were huIgG1_G1m(17). All LC isotypes were huLambda2. .

[0676]

[0677]

[0678] Table 7B. CDR sequences of HC and LC isotypes of 7 of the 9 monoclonal CD3 antibodies from the first batch mentioned above (see table 7B). See Table 3). Sequences were defined according to Kabat. All HC isotypes were huIgG1_G1m(17). All LC isotypes were... huLambda2 .

[0679]

[0680]

[0681] 1-3 Hybridomas that bind to purified T cells from humans and cynomolgus monkeys were selected.

[0682] Design a cell-based binding assay to evaluate the binding of single rat hybridoma supernatant to human ( Figure 2 ) and crab-eating macaques ( Figure 3The binding capacity of purified CD3+ T lymphocytes was assessed. T cells were counted, diluted to 1 × 10^6 cells / mL, and incubated with 0.5 μl / mL of Live / Dead fixative green dead cell stain (Life Technologies, L-2301). Cells were then aliquoted at 100 μl / well (1 × 10^5 cells / well) into U-shaped plates (Falcon 353077). The plates were centrifuged at 300g for 5 min to pellet the cells and remove the supernatant. The plates were briefly vortexed 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 ratio to the lowest concentration of 0.006 μg / mL. The positive control (SP34-2, BD Biosciences 551916) and negative isotype control (mouse IgG1, BD Biosciences 556648) of mouse anti-human CD3 were also diluted to 4.5 μg / mL. 50 μl of each sample was added to 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) at 10 μg / mL was added according to the appropriate species (anti-rat for hybridoma samples, anti-mouse for control antibodies). The plates were incubated at 4°C for 45 minutes and washed twice with staining buffer. 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.). Results were... Figure 2 and Figure 3 As shown in the image.

[0683] In other experiments, purified human T cells were seeded at 1.1 × 10⁵ cells / well in U-shaped plates. The plates were centrifuged at 300 g for 5 min to pellet the cells and remove the supernatant. The plates were briefly vortexed to resuspend the cells. The hybridoma supernatant was diluted to 30 μg / mL in FACS staining buffer (BSA, BD Biosciences 554657), and then serially diluted 11-fold at a 1:3 ratio to a minimum concentration of 0.00017 μg / mL. 50 μl of each sample was added to the T cells and incubated at 4 °C for 1 h. 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 at 4 °C for 1 h and washed twice with staining buffer. The cells were resuspended in 30 μl of FACS buffer and read on a Hypercyte flow cytometer (Intellicyt). Combined with representative dose-response curves of primary human T cell anti-CD3 clones in Figure 5 As shown in the image.

[0684] Representative competitive binding curves with SP34-2 (a commercially available anti-human CD3 antibody with known epitopes and cross-reactivity with cynomolgus monkey CD3) at Figure 6 The results are shown in Table 8. Six clones showed positive binding and also competed with SP34-2 for binding to primary human T cells.

[0685] 1-4 Competitive assays against commercially available CD3 antibodies SP34-2

[0686] The ability of hybridoma supernatant to compete with commercially available anti-human CD3 antibody SP34-2, which has a known epitope and cross-reacts with cynomolgus CD3, was also evaluated. First, a concentration titration curve of AF488-labeled SP34-2 (BD, 557705) was performed to determine a fixed concentration of SP34-2 for the following competitive assay. Briefly, purified human T cells were diluted to 1 × 10^6 cells / mL in PBS. Fc inhibitor (TruStain human Fc inhibitor, Biolegend, 422302) was added at 5 μL / 100 μC, and cells were seeded at 100 μL / well in U-shaped plates. AF488 SP34-2 and an AF488-labeled isotype control (AF488 mouse IgG1, BD, 400129) were serially diluted from 50 μg / mL to 0.049 μg / mL using a 1:2 dilution protocol. Centrifuge the plate at 300xg for 5 minutes to pellet the cells and remove the supernatant. Gently vortex the plate briefly to resuspend the cells. Add 50 μl of each AF488SP34-2 dilution to the cells and incubate at 4°C for 1 hour. Wash the plate 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)). Resuspend the cells in 25 μL of running buffer and read the values ​​on an HTFC screening system (IntelliCyt). Based on the dose-response curve, a fixed concentration of 2 μg / mL SP34-2 was selected for the competitive assay.

[0687] The assay demonstrated that seven hybridomas, which bound to purified T cells, competed with SP34-2 for binding to human T cells. Figure 4 The competitive assay included control antibodies. Unlabeled mouse anti-human CD3, SP34-2 antibodies and mouse anti-human CD3, UCHT1 antibodies were used as positive controls, and rat IgG and mouse isotypes were used as negative controls for AF488-labeled SP34-2. Purified human T cells were diluted in PBS to a concentration of 1 × 10^6 cells / mL. Fc blocking agent (TruStain human Fc blocking agent, Biolegend, 422302) was added to the cells at 5 μL / 100 μL and Live / Dead fixative FarRed dead cell staining agent (Life Technologies L10120) was added to the cells at 0.5 μL / mL, and the cells were incubated at 4°C for 15 minutes. Next, 10 51 × 10⁵ cells / well was aliquoted into 96-well U-shaped plates (Falcon 353077). The plates were centrifuged at 300 x g for 5 min to pellet the cells and remove the supernatant. The plates were gently vortexed briefly to resuspend the cells. The hybridoma supernatant and control antibody were diluted twice to 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. The hybridoma supernatant and control antibody were determined using a 7-point titration with a defined concentration range. Hybridoma supernatant was measured at concentrations from 200 μg / mL to 0.08 μg / mL, and control antibody was measured at concentrations from 100 μg / mL to 0.04 μg / mL. 50 μL of 1X hybridoma supernatant or 1X control antibody containing 2 μg / mL AF488 SP34-2 was added to T cells and incubated at 4°C for 2 hours. 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)). Cells were resuspended in 25 μL of running buffer and read on an HTFC selection system (IntelliCyt). Representative competitive binding curves for SP34-2 (a commercially available anti-human CD3 antibody with a known epitope and cross-reactive with cynomolgus monkey CD3) were obtained. Figure 4 and Figure 6 The results are shown in Table 8. Six clones showed positive binding and also competed with SP34-2 for binding to primary human T cells.

[0688] like Figure 4 As shown, the seven antibodies competed with SP34-2 with similar curves. The rightward shift of the curve relative to the control SP34-2 indicates weaker binding affinity. It can be expected that the isotype control rat IgG will not compete with SP34-2.

[0689] Table 8. Summary of anti-CD3 antibodies binding 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. combine

[0690]

[0691]

[0692] 1-5 screening for hybridoma hits for T cell activation, such as those measured by CD69 upregulation.The ability of hybridomas to hit activated T cells was determined using assays based on primary human and cynomolgus T cells. This was achieved by coating the plates with antibodies to mimic the cross-linking effect of TCR activation. Upon activation, T cells are known to upregulate the surface expression of the protein CD69. This experiment was performed by coating 50 μl of a 10 μg / ml antibody formulation into 96-well plates (Costar#3361) with either 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). The plates were incubated overnight at 4°C. The next day, the plates were washed twice with PBS. Frozen primary T cells (human primary T cells derived from Biological Specialities or Hemacare; cynomolgus primary T cells derived from WorldWidePrimate) were thawed, viability was counted, and the results were compared at 2 × 10⁻⁶. 6 Cells / ml were resuspended in RPMI 1640 medium (Gibco #11875 with 10% HIFBS (Gibco #10062)). 100 μl of cells were added to the plate and incubated overnight (approximately 16 hours) at 37°C and 5% CO2. The next day, the plate was centrifuged at 1300 rpm for 3 minutes to pellet the cells, and the supernatant was discarded. The cells were washed once in PBS and centrifuged as before. 10 μl of a 2.5% solution of Live / Dead Green Fixable Stain (Life Technologies #L23101) in PBS was added to each well and incubated at room temperature in the dark for 10 minutes. Next, 50 μl of a 1% solution of anti-CD69 AF488 (Biolegend #310916, batch number B125271) in FACS buffer (BD Biosciences #554657) was added, and the plate was incubated at 4°C for 45 minutes. Precipitate the cells as described above, discard the supernatant, and resuspend the cells in 150 μl of FACS buffer. Wash the plate twice. After the final wash, resuspend the cells in 150 μl of FACS buffer and read the values ​​on a FACSCanto. 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 measured anti-CD69 staining. Only SP34-2 induced CD69 expression in cynomolgus T cells because it binds to a conserved CD3 sequence region from monkey to human. The OKT3 anti-CD3 clone did not bind to cynomolgus CD3 and did not induce CD69 upregulation. The negative control in both human and cynomolgus T cells was anti-CD20, which was not expressed on T cells. Among the hybridoma clones tested for T cell activation, several induced CD69 expression to a level similar to the positive controls, namely 2B4, 2E6, 3B4, 1F8, 2E9, 3F4, 3G8, 4E5, 5H7, 8B4, 8G8, and 1F1. Except for 5H7, 8B4, and 8G8, most also bound to and activated cynomolgus T cells.

[0693] Framework engineering of 1-6BLW-2E6

[0694] Compared to human immunoglobulin germline sequences, the clones showed high homology and carried frame mutations. Figure 1A and Figure 1B Cloning 2E6 was selected to fit the standard frame sequence. All six mutations on HC and seven mutations on LC were individually or in combination mutated back to the human germline sequence (Table 9). The mutated V region DNA sequence was synthesized and cloned into a mammalian expression vector identical to its parent construct. HC and LC constructs were paired in a matrix to generate proteins carrying single or combined mutations, and protein activity was tested. V48 on LC could not be reverted back to germline. All other reverse mutations were not critical but reduced activity to some extent.

[0695] Table 9. BLW-2E6 Frame Variants .

[0696]

[0697]

[0698] The framework mutation was engineered into a hybridoma clone BLW-2E6, resulting in 80 mutant clones, some of which are shown in Table 10. The binding of these 80 mutant clones to primary human T cells was determined. Figure 7 and Figure 8T cells were counted, diluted to 1 × 10^6 cells / mL, and incubated with 5 μL of Fc inhibitor (TruStain human Fc inhibitor, 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, cells were aliquoted at 100 μL / well (1 × 10^5 cells / well) into 96-well U-shaped plates (Falcon 353077). The plates were centrifuged at 300 x g for 5 min to pellet the cells and remove the supernatant. The plates were gently vortexed briefly to resuspend the cells. The hybridoma supernatant was diluted in FACS staining buffer (BSA, BD Biosciences 554657) to 7.5 μg / mL, 1.5 μg / mL, 0.3 μg / mL, and 0.06 μg / mL. 50 μL of each sample was added to T cells and incubated at 4 °C for 1 hour. Cells were washed once with staining buffer and 50 μL of 5 μg / mL secondary AF647 goat anti-human IgGF(ab')2 (Jackson Immuno Research catalog 109-605-097) was added. The plate was 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)). Cells were resuspended in 25 μL of running buffer and read on an HTFC screening system (IntelliCyt). The results showed that the change in LC at position 48 eliminated binding, preventing further mutation progression. A slight decrease in binding was observed in HC, where all positions reverted to the germline (CD3H231).

[0699] 1-7 C91 scans of BLW-2E6 LC

[0700] Clones 2E6 and their derivatives were poorly expressed, and protein aggregation was observed. A post-translational modification (PTM) risk was predicted for one residue C91 of the light chain, and mutations were made to all other possible amino acids to improve protein stability (Table 10). The mutated V region DNA sequence was synthesized and cloned into a human λ expression vector identical to its parent construct. SPR results showed that changes to valine or leucine at position 91 did not significantly alter binding affinity. This change also bound to the wild-type sequence and the aforementioned 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).

[0701] The sequence information for CD3B376 is provided as follows: :

[0702] CD3H219 HC amino acid sequence (SEQ ID NO:640) :

[0703] QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0704] CD3H219 HC nucleic acid sequence (SEQ ID NO:712)

[0705] caggtgcagctgcagcagtctggccctagactcgtgcggccttcccagaccctgtctctgacctgtgccatctccggcgactccgtgttcaacaacaacgccgcctggtcctggatccggcagagcccttctagaggcctggaatggctgggccggacctactaccggtccaagtggctgtacgactacgccgtgtccgtgaagtcccggatcaccgtgaaccctgacacctcccggaaccagttcaccctgcagctgaactccgtgacccctgaggacaccgccctgtactactgcgccagaggctactcctcctccttcgactattggggccagggcaccctcgtgaccgtgtcctct

[0706] CD3L150 LC amino acid sequence (SEQ ID NO:676) :

[0707] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0708] CD3L150 LC nucleic acid sequence (SEQ ID NO:713) :

[0709] cagtctgctctgacccagcctgcctccgtgtctggctctcccggccagtccatcaccatcagctgtaccggcacctcctccaacatcggcacctacaagttcgtgtcctggtatcagcagcaccccgacaaggcccccaaagtgctgctgtacgaggtgtccaagcggccctctggcgtgtcctccagattctccggctccaagtctggcaacaccgcctccctgaccatcagcggactgcaggctgaggaccaggccgactaccactgtgtgtcctacgctggctctggcaccctgctgtttggcggaggcaccaagctgaccgtgctg

[0710] The VH amino acid sequence of CD3H219 (SEQ ID NO:652) :

[0711] qvqlqqsgprlvrpsqtlsltcaisgdsvfnnnaawswirqspsrglewlgrtyyrskwlydyavsvksritvnpdtsrnqftlqlnsvtpedtalyycargysssfdywgqgtlvtvss

[0712] The VL amino acid sequence of CD3L150 (SEQ ID NO:661) :

[0713] qsaltqpasvsgspgqsitisctgtssnigtykfvswyqqhpdkapkvllyevskrpsgvssrfsgsksgntasltisglqaedqadyhcVsyagsgtllfgggtkltvl

[0714] CD3H219's HCDR1 (SEQ ID NO:662) :NNNAAWS

[0715] CD3H219's HCDR2 (SEQ ID NO:663) :

[0716] RTYYRSKWLYDYAVSVKS

[0717] HCDR3 of CD3H219 (SEQ ID NO:664) :GYSSSFDY

[0718] LCDR1 (SEQ ID NO:671) of CD3L150 :TGTSSNIGTYKFVS

[0719] LCDR2 (SEQ ID NO:673) of CD3L150 :EVSKRPS

[0720] LCDR3 (SEQ ID NO:690) of CD3L150 :VSYAGSGTLL

[0721] The sequence information for CD3B450 is as follows: :

[0722] CD3H231 HC amino acid sequence (SEQ ID NO:675) :

[0723] QVQLQQSGPGLVKPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0724] CD3H231 HC nucleic acid sequence (SEQ ID NO:714) :

[0725] caggtgcagctgcagcagagcggccccggcctggtcaagcccagccagaccctgagcctgacctgcgccatcagcggcgacagcgtgttcaacaacaacgccgcctggtcctggatccgccagagccccagccgcggcctggagtggctgggccgcacctactaccgcagcaagtggctgtacgactacgccgtgtccgtgaagtcccgcatcaccatcaaccccgacaccagcaagaaccagttctccctgcagctgaacagcgtgacccccgaggacaccgccgtgtactactgcgcccgcggctacagcagcagcttcgactactggggccagggcaccctggtcaccgtgtccagc

[0726] CD3L197 LC amino acid sequence (SEQ ID NO:677) :

[0727] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0728] CD3L197 LC nucleic acid sequence (SEQ ID NO:715) :

[0729] Cagtctgctctgacccagcctgcctccgtgtctggctctcccggccagtccatcaccatcagctgtaccggcacctcctccaacatcggcacctacaagttcgtgtcctggtatcagcagcaccccggcaaggcccccaaagtgatgatctacgaggtgtccaagcggccctccggcgtgtccaacagattctccggctccaagtccggcaacaccgcctccctgacaatcagcggactgcaggccgaggacgaggccgactactactgtgtgtcctacgccggctctggcaccctgctgtttggcggcggaacaaagctgaccgtgctg

[0730] The VH amino acid sequence of CD3H231 (SEQ ID NO:657) :

[0731] qvqlqqsgpglvkpsqtlsltcaisgdsvfnnnaawswirqspsrglewlgrtyyrskwlydyavsvksritinpdtsknqfslqlnsvtpedtavyycargysssfdywgqgtlvtvss

[0732] The VL amino acid sequence of CD3L197 (SEQ ID NO:678) :

[0733] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCVSYAGSGTLLFGGGTKLTVL

[0734] CD3H231's HCDR1 (SEQ ID NO:662) :NNNAAWS

[0735] CD3H231's HCDR2 (SEQ ID NO:663) :

[0736] RTYYRSKWLYDYAVSVKS

[0737] CD3H231's HCDR3 (SEQ ID NO:664) :GYSSSFDY

[0738] LCDR1 (SEQ ID NO:671) of CD3L197 :TGTSSNIGTYKFVS

[0739] LCDR2 of CD3L197 (SEQ ID NO:673) :EVSKRPS

[0740] LCDR3 of CD3L197 (SEQ ID NO:690) :VSYAGSGTLL

[0741] Table 10. Engineered variants of BLW-2E6 LC as measured by C91 scan

[0742] C91 scan Peptide ID CD3 2E6 LC CD3L124 CD3 2E6 LC, C91S CD3L146 CD3 2E6 LC, C91G CD3L147 CD3 2E6 LC, C91E CD3L148 CD3 2E6 LC, C91D CD3L149 CD3 2E6 LC, C91V CD3L150 CD3 2E6 LC, C91A CD3L151 CD3 2E6 LC, C91R CD3L152 CD3 2E6 LC, C91K CD3L153 CD3 2E6 LC, C91N CD3L154 CD3 2E6 LC, C91M CD3L155 CD3 2E6 LC, C91I CD3L156 CD3 2E6 LC, C91T CD3L157 CD3 2E6 LC, C91W CD3L158 CD3 2E6 LC, C91Y CD3L159 CD3 2E6 LC, C91L CD3L160 CD3 2E6 LC, C91F CD3L161 CD3 2E6 LC, C91Q CD3L162 CD3 2E6 LC, C91H CD3L163 CD3 2E6 LC, C91P CD3L164

[0743] 1-8 Binding of BLW-2E6 CD3 mAb to hCD3ε construct as measured by ProteOn SPR

[0744] The 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 (manufactured by Janssen, referred to as hCD3ε(1-27)-Tn25) was measured using ProteOn SPR (Bio-Rad). Goat anti-human FcIgG (Jackson Immunoresearch, catalog 109-005-098) was directly immobilized at 30 μg / mL in acetate buffer (pH 5.0) on all six ligand channels of a GLC sensor chip (Bio-Rad, catalog 176-5011) at a vertically oriented flow rate of 30 μL / min in PBS containing 0.005% Tween-20. The immobilization density averaged approximately 6000 response units (RU), with a variation of less than 5% between different channels. Five different mAbs with vertical ligand orientation were captured on the anti-human FcIgG 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 5-fold 3-fold dilution series, 1 μM hCD3ε(1-27)-Tn25 was flow-in as the analyte, binding to the captured mAbs with 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 min at a flow rate of 100 μL / min. The binding surface was regenerated using an 18-second pulse of 0.8% phosphate for the next interaction cycle to remove the antigen and bound mAbs. The raw data was processed by subtracting two sets of reference data from the response data: 1) the inter-point signal to correct for non-specific interactions between Ag and the empty chip surface; and 2) the buffer channel signal to correct for baseline drift due to dissociation of the captured mAb surface over time. The treatment data for each mAb at all concentrations were globally fitted to a simple 1:1 Langmuir binding model to extract kinetics (k... on k off ) and affinity (K D Estimates of the constant. Results are provided in Figure 11 middle.

[0745] Table 11. Summary of kinetics / affinity for binding of BLW-2E6 variants to hCD3ε(1-27)-Tn25 (N=1)

[0746]

[0747] N / A = Unspecified

[0748] 1-9 Binding of anti-CD3 monoclonal antibodies to human T cells

[0749] After hybridization with the antigen-specific target arm, the in vitro binding affinity of CD3B376 and CD3B450 to human T cells was determined by flow cytometry. Preliminary studies on human T cells were conducted to determine the saturation binding constant (KdT) of the anti-CD3 tracer molecule. Then, a competitive binding assay was performed using a fixed concentration of the tracer ([T]) and a titrated concentration of the test mAb. The IC50 value (the concentration at which 50% inhibition is achieved) of the test molecule was used to determine the binding affinity (K) using the following formula. d ):K d =IC50 / (1+([T] / K) d T). The saturation binding constant (K2) of the tracer, commercially available Alexa Fluor 488 SP34-2 anti-CD3 (BioScience #557705) (data not shown), was determined using five human donors. d T).

[0750] Determine the saturation binding constant (K) of the tracer. d T)

[0751] Methods: Human PanT cells were cryopreserved in a nitrogen atmosphere until use. T cells were thawed, washed with PBS, resuspended in FACS staining buffer, counted (stating viability), and resuspended at 0.5 × 10^6 cells / mL. FarRedLive / Dead staining agent (Life Technologies, AKA Invitrogen #L34974) (50 μL DMSO to a vial) was added at 1 μL / 1 × 10^6 cells; and FcR blocking agent (Miltenyi Biotec #130-059-901) (1 mL 1:20 dilution / 0.5 × 10^6 cells) was added to the cells, with each addition for 10 minutes. Cells were seeded at 50,000 cells / well and washed. Incremental concentrations of Alexa Fluor 488SP-34 anti-CD3 were added to the T cells, and the cells were incubated at 4°C for 2 hours. Wash the cells to remove unbound antibodies, fix for 15 minutes, wash again, and resuspend in FACS staining buffer containing 1 mM EDTA.

[0752] Binding was measured using an iQue Intellicyte flow cytometer. Gating was performed on T cell populations, then on single cells, and finally on live cells (FL4). The geometric mean of staining was determined for each well (FL1).

[0753] The average fluorescence intensity values ​​were plotted as a function of antibody molecule concentration and analyzed using Prism software in unit site binding analysis (total binding). Figure 9 The software calculates the corresponding K. dThe value describes the binding of antibody molecules to their receptor (CD3 on human PanT cells), a binding that follows the law of mass action. The formula is as follows: Y = (B max ×X) / (K d +X); where: Bmax is the maximum associativity; K d It is the ligand concentration required to achieve half-maximal binding.

[0754] Results: K for each donor was derived. d The values ​​were calculated, and the average value was obtained. The saturation binding constant (Ks) for human T cells was derived. d T) is 5.6 ± 1.0nM (n = 4), and is used in the aforementioned formula to determine K. d Combined with affinity.

[0755] The binding affinity of anti-CD3 mAbs was determined by a competitive assay.

[0756] Methods: Competitive binding studies were conducted using bivalent anti-CD3 antibodies.

[0757] • Anti-CD3 divalent: CD3B376 and CD3B450 ( Figure 10 )

[0758] Human PanT cells were used to determine the binding affinity of the test mAb. The tracer used was commercially available Alexa Fluor 488SP-34 anti-CD3 (BioScience #557705), and the saturation binding constant of this tracer is as described above.

[0759] T cells were cryopreserved in a nitrogen tank until use. After thawing, the T cells were washed with PBS, resuspended in FACS staining buffer, counted, and viability was noted. The results were then plotted at 0.5 × 10⁻⁶. 6 Resuspend cells at 100 cells / mL. Add 1 μL of FarRedLive / Dead staining agent (Life Technologies, AKA Invitrogen #L34974) (50 μL of DMSO to a vial) to 1 × 10^6 cells; and add FcR blocking agent (Miltenyi Biotec #130-059-901) (1 mL of 1:20 dilution / 0.5 × 10^6 cells) to the cells, 10 minutes each. Seed cells at 50,000 cells / well and wash.

[0760] The mAb (and isotype control) were serially diluted 1:2 from initial concentrations of 1000 μg / mL or 200 μg / mL (2X), and a fixed concentration of tracer (5 μg / mL; 2X) was mixed together to obtain a 1X concentration. Therefore, the final (1X) concentration of the tracer was 2.5 μg / mL = 16.6 nM. This mixture was added to T cells and incubated at 4°C for 2 hours. Cells were then washed to remove unbound antibodies, fixed for 15 minutes, washed again, and resuspended in FACS staining buffer containing 1 mM EDTA.

[0761] Binding was measured using an iQue Intellicyte flow cytometer. Gating was performed on T cell populations, followed by single cells, and then live cells (FL4). The geometric mean of staining was measured for each well (FL1). The acquired average fluorescence intensity values ​​were plotted as a function of log antibody molecule concentration (converted to nM) and analyzed using Prism software in an S-shaped dose-response (variable slope) to derive EC50 / IC50 values ​​(in nM). Binding affinity (Kb) was derived using the following formula. d ):K d =IC50 / (1+([T] / K) d T)). Where: K d [T] represents the affinity of the competitor (unlabeled molecule); [T] represents the IC50 of the test compound (in nM); [T] represents the concentration of the tracer (16.6 nM); K d T is the K of the tracer determined by saturated binding. d (For human beings, 5.6 nM).

[0762] Production of monoclonal and bispecific antibodies

[0763] The bispecific CD3 antibody of the present invention can be generated via controlled Fab arm exchange (FAE), as described in the following references: Labrijn et al., 2013, PNAS, Vol. 110, No. 13: pp. 5145-5150; PCT Publication WO2011 / 131746; or Labrijn et al., 2014, NatProtoc, Vol. 9, No. 10: pp. 2450-2463. In short, in this in vitro method, two full-length parental bivalent antibodies are provided, each containing a mutation in its CH3 region that favors heterodimer formation, thereby generating a bispecific antibody containing a half-arm from each parental antibody. Mutations that can be used to promote heterodimer formation are, for IgG1 antibodies, F405L from one parental antibody and R409K from the other, or for IgG4 antibodies, F405L and K409R from one parental antibody while retaining wild-type CH3.

[0764] Monospecific antibodies are expressed in the HEK cell line under the action of the CMV promoter.

[0765] Parental antibodies were purified using a Protein A column containing elution buffer of 100 mM NaAc (pH 3.5) and 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.

[0766] After purification, the parental antibody was mixed in 75 mM cysteine-HCl under reducing conditions and incubated at 31 °C for 4 h. The recombinant reaction was based on a molar ratio in which a predetermined amount of target parent (e.g., 10 mg or approximately 71.8 nanomoles) was combined with CD3 antibody (e.g., approximately 67.8 nanomoles), wherein the amount of target parent antibody added was 6% higher than that of CD3 antibody. The recombinant was then dialyzed against PBS to remove the reducing agent. A bispecific antibody reaction was performed with an excess of target parent antibody (ratio) to minimize the amount of unreacted CD3 parent antibody remaining after recombinant reaction. After partial reduction of the parental mAb, the reducing agent was removed by overnight dialyzing into PBS.

[0767] result: Figure 10 The inhibition curves of the bivalent and monovalent anti-CD3 constructs CD3B376 and CD3B450 competitively binding to the AlexaFluor488SP-34 anti-CD3 tracer antibody are shown. Increasing the concentration of the tested anti-CD3 antibody reduced the binding of the AlexaFluor488 tracer antibody, thereby reducing the mean fluorescence intensity (MFI). IC50 values ​​were generated, and K was calculated using the aforementioned formula. d Affinity is summarized in Table 13.

[0768] The CD3B376 binding site is more tightly bound than both the bivalent and monovalent forms of CD3B450.

[0769] Table 13. IC50 and K0 values ​​of anti-CD3 divalent and monovalent CD3B376 and CD3B450 constructs d Affinity value

[0770]

[0771] 1-10 Conformational stability of anti-CD3 monoclonal antibodies

[0772] The conformational stabilities of anti-CD3 antibodies CD3B376, CD3B389 (IgG1σ form of CD3B376; heavy chain is SEQ ID NO: 729; light chain is SEQ ID NO: 676), CD3B450, and CD3B467 (IgG1σ form 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.

[0773] 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 show some differences in thermal stability. The CD3B376 (IgG4PAA) molecule shows 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 (IgG4PAA) molecule shows 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 shows 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 shows 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.

[0774] Table 14. Summary of thermal conversion data for anti-CD3 antibodies in PBS. Values ​​represent the average of repeated runs. HC peptides are provided. The sequence information of the LC peptide (SEQ ID NO in parentheses).

[0775]

[0776] The two IgG1 antibodies show lower stability compared to the corresponding IgG4PAA 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).

[0777] Crystal structure of CD3B334 Fab complexed with CD3eN-terminal peptide 1-11

[0778] The anti-CD3mAb CD3B334 (CD3H231 / CD3L137) was modified to increase the "humanity" index by replacing multiple framework residues in the antibody with human-derived residues. This procedure produced antibody CD3B334 with the following mutations: D43G / L49M / L50I / S62N / Q85E / H89Y in VL compared to the parental VLCD3L124; and R10G / R13K / V73I / R79K / T83S / L96V in VH compared to the parental VHCD3H219. The His-tagged Fab fragment of CD3B334 was expressed in HEK293Expi cells and purified using affinity chromatography and size exclusion chromatography. The N-terminal 9-meric peptide of human CD3e was synthesized using New England Peptide (lot number V1108-19 / 21) and mixed with Fab at a molar ratio of 10:1 (excess peptide). The complex was crystallized from a solution containing 4M sodium formate in 0.1M Tris (pH 8.5) by vapor diffusion. The crystals belong to the orthorhombic crystal system, space group P212121, with a cell size of [missing information]. There is a composite molecule within the asymmetric unit. Using the crystal structure of Fab as the search model, molecular substitution is employed to... The resolution determines the structure of the complex.

[0779] CD3B334 binds to residues 1-6 of CD3e. The N-terminal Gln of the peptide is in the form of pyroglutamate and is located in a hydrophobic environment, between F107 of HCDR3 and L99 of LCDR3. Two arginine residues, R52 and R56, from HCDR2 interact electrostatically with acidic residues of CD3. A total of 16 residues form the complementary site of CD3B334. Residues from all CDRs except LCDR2 are in direct contact with the CD3 peptide (in... (within distance) (see) Figure 18 ).

[0780] 2 PSMA antibody

[0781] 2-1 Generation of PSMA cell lines

[0782] 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) were generated for use as screening tools, thereby evaluating anti-PSMA leads using standard molecular biology techniques and internal expression vectors with CMV promoters. The vectors were transiently transfected into suspension HEK293F cells using standard methods. Transfected 293F suspension cells were seeded in serum-added growth medium to allow them to adhere and select for stable plasmid integration. Single cell populations were selected by serial dilution, and the expression of PSMA surface receptors was quantified by FACS using rabbit polyclonal antibody (OAAB02483, Aviva Systems Biology) affinity-purified with PSMAL antibody (Center) as the primary antibody, and R-PE anti-rabbit secondary antibody (111-116-144, Jackson Immuno Research Laboratories, Inc.) and rabbit polyclonal IgG (SC-532, Santa Cruz Biotechnology) as isotype controls.

[0783] Human PSMA-expressing cell lines were generated using a lentivirus (Genecopoeia, catalog number EX-G0050-Lv105-10) containing full-length human PSMA (FOLH1_HUMAN, SEQ ID NO:51) and puromycin for selecting PSMA-positive cells. PSMA-negative HEK293F cells (ATCC) were transduced with lentiviral particles to overexpress human PSMA. After transduction, cells expressing both PSMA and the resistance marker were selected by treating the confluent cells, which were then grown in DMEM + 10% HIFBS (Life Technologies) and supplemented with varying concentrations of puromycin (Life Technologies).

[0784] In addition to the HEK-derived cell line, 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 derived from LNCaPFGC cells that had grown in vivo and metastasized to the bone marrow (Thalmann et al., 1994, Cancer Research, Vol. 54, pp. 2577-2581).

[0785] 2-2 Production of soluble PSMA ECD protein

[0786] Recombinant chimpanzee PSMA extracellular domain (ECD) protein (chimpanzee PSMA ECD, SEQ ID NO:52) was generated for panning, and the anti-PSMA lead was evaluated using standard molecular biology techniques and an internal expression vector with a CMV promoter. A chimpanzee PSMAECD gene fragment (amino acids 44–750 of SEQ ID NO:49) containing 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 techniques (US Patent 6,670,127; US 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, GE Healthcare), with the elution buffer being a Dulbecco phosphate-buffered saline (Thermofisher, #14040) containing 0.5 mM ZnCl2 and calcium and magnesium to stabilize PSMA dimerization. Fractions containing the protein of interest were pooled, and protein concentration was determined by A280. This material was used for binding and affinity measurements and is referred to as PSMG8.

[0787] Chimpanzee PSMAECD was also biotinylated for panning. BirA plasmids containing Avi-tagged proteins, co-transfected into mammalian cells, were 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 the ER-retaining 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 controlled by the CMV promoter. To generate the biotinylated PSMA antigen, PSMA plasmid DNA was added to the BirA plasmid at a 4-fold excess (w / w) and then to the transfection mixture.

[0788] Biotinylation of the chimpanzee PSMAECD protein was performed via Avi tagging and co-transfection with a BirA expression construct. The resulting secreted protein was purified using a two-step purification process: 1) IMAC purification using a HisTrapHP column (GE Healthcare), and 2) size exclusion purification (Superdex 200, GE Healthcare), with the elution buffer being a Dulbecco phosphate-buffered saline (Thermofisher, #14040) containing 0.5 mM ZnCl2 and calcium and magnesium to stabilize PSMA dimerization. Endotoxin levels in the protein were tested prior to use in phage panning studies.

[0789] A recombinant cynomolgus monkey PSMA extracellular domain (ECD) protein (cynomolgus monkey PSMAECD, SEQ ID NO:53) was cloned and expressed as previously described for chimpanzee PSMAECD. This extracellular domain protein corresponds to amino acids 44-750 of SEQ ID NO:50 and possesses 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 the cynomolgus monkey PSMAECD protein was performed via the Avi tag and co-transfection with a BirA expression construct, and the resulting secreted protein was purified using a two-step purification process using an IMACHisTrapHP column (GE Healthcare) and a MonoAvidin column. Endotoxin content 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.

[0790] A second recombinant cynomolgus monkey PSMAECD protein (cynomolgus monkey PSMAFc, SEQ ID NO: 54) containing IgG1Fc (SEQ ID NO: 593) was cloned and expressed using standard molecular biology techniques and an internal expression vector with a CMV promoter. The cynomolgus monkey PSMAFc protein was transiently expressed in HEK293 Expi cells. Five days after transfection, transient transfection of PSMG3 in HEK293 Expi cells was harvested, clarified by centrifugation (30 min, 6000 rpm), and filtered (0.2 μPES membrane, Corning). The relative amounts of IgG were determined using an Octet instrument (ForteBio) and purified known IgG (isotype) incorporated into the used culture medium to generate a standard curve.

[0791] Clarified cynomolgus monkey PSMAFc 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 eluted with 10 column volumes of 0.1 M sodium acetate (pH 3.5). Peak fractions were combined, neutralized with 2 M Tris (pH 7), and filtered (0.2 μL). The neutralized protein sample was dialyzed overnight at 4°C through three variations of dPBS containing Ca2+, Mg2+, and 0.5 mM ZnCl2 (pH 7.2). The next day, the sample was removed from the dialysate, filtered (0.2 μL), and the protein concentration was determined by absorbance at 280 nm using a BioTek SynergyHT™ spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size-exclusion HPLC (Dionex HPLC system). Endotoxin levels were measured using the LAL assay (Pyrotell-T, Associates of Cape Cod). The purified protein was stored at 4°C.

[0792] The recombinant human PSMA extracellular domain (ECD) protein (human PSMA ECD, SEQ ID NO:55) was cloned, expressed and purified as previously described for the PSMA ECD protein in chimpanzees and cynomolgus monkeys. This extracellular domain protein corresponds to amino acids 44-750 of SEQ ID NO:51 and has an N-terminal Avi tag and a 6His tag (SEQ ID NO:596).

[0793] 2-3 Identification of anti-chimpanzee and anti-human PSMAFab

[0794] Panning with recombinant proteins

[0795] First solution panning of de novo human Fab-pIX libraries consisting of VH1-69, 3-23, and 5-51 heavy chain libraries paired with libraries 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] An alternating panning method was used, wherein one round of phage capture was performed according to the manufacturer's instructions on streptavidin magnetic beads (Invitrogen, catalog 112.05D, lot 62992920) coated with biotinylated chimpanzee PSMA ECD, followed by phage capture on ProtG beads (Invitrogen, catalog 10003D) coated with cynomolgus monkey PSMA-Fc, and then on Sera-magDoubleSpeed ​​neutral avidin magnetic beads (Thermo, catalog 7815-2104-011150) coated with biotinylated chimpanzee PSMA ECD. This panning yielded two hits: PSMM18 and PSMM25.

[0796] Whole-cell panning of anti-PSMA Fab

[0797] Additional panning experiments were performed on whole cells using either the first-round output from the chimpanzee ECD panning experiments described above or a fresh de novo phage library as input. Briefly, according to standard protocols known in the art, phages were generated via helper phage infection and concentrated via PEG / NaCl precipitation. The phage library was pre-cleared by gently shaking overnight at 4°C on untransfected parental HEK293F cells. Following PEG / NaCl precipitation, the pre-cleared library was incubated with either chimpanzee PSMA-expressing HEK293 cells or LNCAP cells with gentle shaking at 4°C for 2 hours. Unbound phages were removed and phage-bound cells were recovered via a Ficoll gradient, and after several washing steps, cells carrying bound phages were incubated with 1 mL of TG-1 *E. coli* culture at 37°C for 30 minutes without stirring. The resulting mixture was inoculated onto LB-carbenicillin-1% glucose plates and grown overnight at 37°C. Then repeat the process for subsequent rounds of selection.

[0798] bacteriophage Fab-pIX was converted to Fab-His to produce E. coli supernatant.

[0799] The resulting phage Fab-pIX hit was converted to Fab-His using a standard procedure. Plasmid DNA was isolated from phage-panned *E. coli* (Plasmid Plus Maxi kit, Qiagen catalog 12963) 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 purified by gel electrophoresis (MinElute PCR purification kit, Qiagen catalog 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 *E. coli* strain TG-1 and cloned. Fab-His supernatant was produced from the clone by overnight induction of the culture with 1 mM IPTG. After centrifugation of the overnight culture, the clarified supernatant was prepared for use in downstream assays. To determine the relative expression levels of different Fab-his supernatants, serially diluted supernatants were subjected to an anti-k (Southern Biotech, catalog number 2061-05) ELISA. All clones tested showed similar Fab-his expression (data not shown).

[0800] Cell binding of Fab-his fusion from E. coli

[0801] Cell-based binding assays were designed to evaluate the binding affinity of individual Fab-his fusions from *E. coli* supernatant to PSMA-expressing cells. Following pIX excision, individual Fab clones were isolated from the third round output of all panning experiments. Binding of Fab clones to HEK cells expressing chimpanzee and cynomolgus monkey PSMA, as well as to human PSMA on LNCaP cells, was tested. Briefly, PSMA-expressing cells were aliquoted at a density of 200,000 / well into V-shaped plates (CoStar 3357) and incubated on ice for 1 hour with 100 μl of supernatant expressing the Fab fragment. Cells were washed twice with PBS containing 2% FBS and stained on ice for 1 hour with mouse anti-human κ-RPE antibody (Life Technologies, catalog MH10514). Cells were washed twice with PBS containing 2% FBS and resuspended in 100 μL of the same wash buffer. Plates were read using a BD FACSArray flow cytometer. Healthy cell populations were gated in real-time using forward and side scattering, and PE staining of cells within the gate was analyzed. FACS data were analyzed in FlowJo software. Mean fluorescence intensity (MFI) was calculated and exported to Microsoft Excel. Fab clones showing binding ≥3-fold background and not showing binding with the HEK293 cell line in all three PSMA species (cynomolgus monkey, chimpanzee, and human) were marked as “preliminary positive.” Fab clones were sequenced and moved forward to clone into mammalian expression vectors for rescreening. True positives were selected from the binding of Fab supernatant expressed in mammalian cells to PSMA-expressing cell lines.

[0802] Preparation of mammalian Fab

[0803] To convert *E. coli* Fab into mammalian expression Fab, In-FusionHD cloning (ClonTech catalog number 638918) was used according to the manufacturer's protocol. In short, the nucleotide sequences of clones that had passed initial screening and been transferred into mammalian Fab form were loaded into the "InFu Primer Finder v1.2.3" program (in-house developed software), which generates a list of isotype-specific PCR primers for producing PCR fragments for seamless cloning into the huKappa_muIgGSP and huG1Fab expression vectors. These vectors are in-house vectors with a CMV promoter based on pcDNA3.1. Following the seamless cloning process, *E. coli* clones were isolated using standard protocols, sequenced, and transfected into HEK293 cells. Mammalian PSMAFab was prepared to confirm binding to cell lines expressing PSMA by harvesting 20 ml of supernatant from the transfection after 5 days.

[0804] Rescreening of hits from whole-cell panning in mammalian supernatant form

[0805] The mammalian expression of Fab supernatant was confirmed using the aforementioned whole-cell binding assay. Binding of Fab to human PSMA (LNCaP), chimpanzee, and cynomolgus monkey cells was tested, and reverse screening was performed for cases that did not bind to the parental HEK cell line. Table 19 shows the hit characteristics of mammalian Fab supernatant binding to PSMA-expressing cells. Many hits from E. coli supernatant could not be confirmed by the mammalian protein expression. PSMM48 showed high binding to cynomolgus monkey PSMA-expressing cells and some binding to chimpanzee PSMA-expressing cells, 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 or cynomolgus monkey PSMA. Mammalian Fab supernatant PSMM52, M56, and M57 bound to all three cell lines. PSMM50, M51, and M54 showed more binding to chimpanzee or cynomolgus monkey cells. M58 shows a slight hybridization between chimpanzees and cynomolgus monkeys.

[0806] Table 19. Mammalian Fabry Diseases Binding to PSMA-Expressing Cells as Measured by Geo-MFI (Mean Fluorescence Intensity) Protein hit characteristics

[0807]

[0808]

[0809]

[0810]

[0811] Dose-response curve of Fab expressed in mammals

[0812] Once the mammalian-expressed Fab clone was confirmed to have a positive binding to PSMA-expressing cell lines as pure Fab supernatant, the supernatant was normalized to protein concentration using Octet or protein gel, and dose-response curves were generated using the previously described protocol to confirm PSMA binding. Figures 22 through 24 show titration curves demonstrating hits with all three PSMA-expressing cell lines. Figure 22A , Figure 22B , Figure 22C and Figure 22D The titration curve of anti-PSMA panning hits relative to LNCaP cells is shown. Figure 23A , Figure 23B , Figure 23C and Figure 23DThe titration curve of anti-PSMA panning hit relative to chimpanzee PSMAHEK cells is shown. Figure 24A , Figure 24B , Figure 24C and Figure 24D Titration curves of anti-PSMA panning hits relative to cynomolgus monkey PSMAHEK cells are shown. Binding characteristics of the hits were compared between cell lines expressing PSMA from different species. PSMM52 supernatant was used as a positive control throughout the experiment. The binding of some hits to PSMA-negative parental HEK cell lines or the lack thereof to PSMA-positive cell lines was reduced due to the N-linked glycosylation site in the CDR. Of the remaining 11 Fab hits, 10 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 to bispecific antibody form to test T-cell retargeting killing of PSMA-positive targets. The panning antigen for each hit is shown in Table 20.

[0813] Table 20. Antigens Hit by Each Selection

[0814]

[0815] Preparation of anti-PSMA mAb

[0816] A total of 12 clones exhibiting binding to cells expressing all three PSMA species were ultimately converted into mAbIgG4 with Fc substitutions for the S228P, F234A, and L235A (PAA) isotypes via restriction cloning. Briefly, constructs corresponding to the Fab clones that passed initial screening were digested with HindIII and ApaI. The gel-purified fragments were ligated into expression vectors containing the CMV promoter vDR000215, and these CMV-driven expression vectors contained human IgG4-PAAFc for complete mAb expression. This allowed for rapid production of bispecific antibodies. The heavy and light chains of each PSMA mAb were expressed using the previously described expression vectors, with both vectors transiently co-transfected into 293Expi or CHO cell lines for mAb expression. The CDR sequences of the cross-species positive PSMA Fabs generated by phage panning are shown in Table 21 below. The VH and VL sequences of the selected Fabs are shown in Table 22 below. The heavy chain and light chain sequences of mAbs generated by Fab are shown in Table 23 below.

[0817] Table 21. CDR sequences of FAbs from phage panning (according to Kabat definition) (corresponding SEQ ID NO listed in...) (in parentheses)

[0818]

[0819]

[0820]

[0821] Table 22. VH and VL sequences of PSMA Fab

[0822]

[0823]

[0824]

[0825] Table 23 Heavy chain and light chain sequences of PSMA monoclonal antibodies with corresponding SEQ ID NO.

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833] A monospecific anti-PSMA antibody, PSMB119, was generated, comprising VH and VL regions having the VH region of SEQ ID NO:130 and the VL region of SEQ ID NO:131, and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB120, was generated, comprising VH and VL regions having the VH region of SEQ ID NO:128 and the VL region of SEQ ID NO:129, and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB121, was generated, comprising VH and VL regions having the VH region of SEQ ID NO:126 and the VL region of SEQ ID NO:127, and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB122, was generated, comprising a VH region having the VH region of SEQ ID NO:125 and a VL region having the VL region of SEQ ID NO:111, and an IgG4 constant region having S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB123, was generated, comprising a VH region having the VH region of SEQ ID NO:123 and a VL region having the VL region of SEQ ID NO:124, and an IgG4 constant region having S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB124, was generated, comprising a VH region having the VH region of SEQ ID NO:121 and a VL region having the VL region of SEQ ID NO:122, and an IgG4 constant region having S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB126, was generated, comprising VH and VL regions having VH of SEQ ID NO:118 and VL of SEQ ID NO:119, and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB127, was generated, comprising VH and VL regions having VH of SEQ ID NO:116 and VL of SEQ ID NO:117, and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB128, was generated, comprising VH and VL regions having VH of SEQ ID NO:114 and VL of SEQ ID NO:115, and an IgG4 constant region with S228P, F234A, and L235A substitutions.A monospecific anti-PSMA antibody, PSMB129, was generated, comprising VH and VL regions having VH of SEQ ID NO:110 and VL of SEQ ID NO:111, and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody, PSMB130, was generated, comprising VH and VL regions having VH of SEQ ID NO:112 and VL of SEQ ID NO:113, and an IgG4 constant region with S228P, F234A, and L235A substitutions.

[0834] 2-5 Crystal structure of human PSMA ECD bound to anti-PSMA Fab PSMB83 (also known as PSMM84)

[0835] PSMA is a homodimeric protein expressed on the cell surface. PSMA is a type II integrative glycoprotein with 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. To better understand the binding site between PSMA and the antibody, the extracellular region (ECD) of human PSMA was determined at resolution.

[0836] The extracellular region of human PSMA (residues 44-750) is located in the HighFive region with an N-terminal gp67 signal peptide and a subsequently cleavable six-histidine tag (SEQ ID NO: 596). TM Expression in insect cells. The secreted protein was purified from the supernatant via a three-step procedure, which 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 rapidly frozen in liquid nitrogen and stored at -80°C in 10 mM HEPES (pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.1 mM ZnCl2).

[0837] 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 hexahistine 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 and 20 mM Tris (pH 7.4).

[0838] The human PSMA ECD / PSMB83Fab complex was prepared via a three-step procedure. First, the Fab buffer was exchanged into 20 mM MES (pH 6.0, 150 mM NaCl). Then, Fab and PSMA were mixed (1.5 moles of Fab relative to the 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 monoS5 / 50 column in 20 mM MES (pH 6.0) and eluted with a NaCl gradient.

[0839] Crystals suitable for X-ray diffraction were obtained at 20 °C using a seated drop vapor diffusion method and a Mosquito LCP robot (TTP Labtech). PSMB83Fab crystals bound to human PSMAECD were grown from 18% PEG 3kDa, 0.2M (NH4)2SO4, 0.1M Tris (pH 8.5) using microseeds and an initial PSMA / Fab complex concentration of 7.3 mg / mL. Crystals of free PSMB83Fab were obtained from 25% PEG 3kDa, 0.2M LiCl, 0.1M acetate (pH 4.5), with an initial Fab concentration of 8.8 mg / mL.

[0840] The structure was resolved using molecular substitution (MR) with Phaser (Phaser crystallography software, University of Cambridge). The MR search model for the PSMB83 (also known as PSMM84) Fab structure is PDB code 4M6O. PSMA (PDB code: 2C6G) and PSMB83 Fab (…) were used to resolve the structure. The PSMA / Fab complex structure was resolved using a MR search model (resolution structure; data not shown). The structure was refined using PHENIX (Adams et al., 2004) and modeled using COOT (Emsley and Cowtan, 2004). All other crystallographic calculations were performed using the CCP4 suite (Collaborative Computational Project Number 4, 1994). All molecular graphics were rendered using PyMol (PyMOL Molecular Graphics System, version 1.4.1). The LLC. is generated, and the Complementary Determinant Region (CDR) is determined using the Kabat definition.

[0841] The PSMA / Fab structure comprises Fab light chain residues 1-211, Fab heavy chain residues 1-224 (excluding disordered residues 138-146), and PSMA residues 56-750, corresponding to protease (residues 56-116 and 352-590), apical (residues 117-351), and helical (residues 591-750) domains, and seven of the ten possible N-linked glycans (Asn-76, -121, -140, -195, -459, -476, and -638) in each PSMA dimer subunit. The PSMA active site is located at the interface between these three domains and contains two zinc atoms coordinated by histidine (H377 and H553) and glutamic acid / aspartic acid (D387, E424, E425, and D453) residues and water molecules. The asymmetric unit cell contains a PSMA dimer, where each subunit is bonded in a manner similar to that of PSMB83Fab. The Fab / PSMA binding site is precisely defined by an electron density map, which allows for reliable localization of the binding residues. The Fab and PSMA molecules are... Figures 25 to 30 Numbered sequentially.

[0842] PSMB83 epitopes, complementary sites, and interactions. PSMB83 is the parental anti-PSMA Fab arm in the bispecific antibody PS3B27, recognizing conformational epitopes and discontinuous epitopes in the apical domain of PSMA. Figure 25 The surface area of ​​the PSMA embedded in the Fab is approximately... Specifically, the PSMB83 epitope residues are I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326. Helical α7 (residues Y299-E307) is the general region of the epitope and binds to both the Fab heavy chain and the light chain CDR. At one end of the helix, Y299 and Y300 are associated with Fab residue Y57. H W94 L It forms an aromatic cluster with PSMA residues F235 and P237, while at the other end of the helix, E307 interacts with R91. L and hydrogen bond Y32 L A salt bridge is formed. Figure 26 and Figure 27 The major interactions between PSMA and the light and heavy chains of PSMB83 are shown. The PSMB83 epitope residues are conserved between humans and cynomolgus monkeys. Figure 28 Furthermore, the bispecific antibody PS3B27 was shown to bind to human and cynomolgus monkey PSMA with similar affinity. In contrast, the human-to-mouse G238A mutation, particularly 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 affinity of N59...H hydrogen bond contact and with W94 L The π-stacking interaction.

[0843] The complementary site of PSMB83 is composed of residues from all CDRs except CDR-L2 and CDR-H1. Figure 29 Specifically, the complementary residue is the 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 The interaction contact between PSMA and PSMB83 is illustrated. The accessible location of the epitope favors the binding of the PSMB83 Fab arm of the PS3B27 bispecific antibody to membrane-bound PSMA, 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 remotely to the active site and does not induce any significant structural changes in PSMA that could affect enzyme function, such as loop movement closing the active site or substitution of catalytic residues (RMSD for Cα superposition of PSMA molecules in Fab-bound and Fab-unbound structures is ). (Barinka et al., 2007)).

[0844] 2-6 Anti-PSMA affinity maturation

[0845] Affinity maturation was performed on anti-PSMAFab phage clones from two PSMA affinity maturation libraries to identify antibodies with increased binding affinity compared to the parent PSMB127 (fabID = PSMB83, also known as PSMM84). Two libraries were generated for affinity maturation of PSMB127. In the first library, heavy chain CDR1 and CDR2 were randomized according to the design (PH9H9L1) in Table 24. The H-CDR3 fragment was amplified by PCR from pDR000024032 and digested with SacII+XhoI. This fragment was cloned into the PH9H9L1 / PH9L3 library. It was transformed into E. coli MC1061F' cells, and phages displaying this Fab library were generated. In the second library, light chain CDRs were randomized according to the design (PH9L3L3) in Table 25. The heavy chain from PSMB83 (PSMH360) was amplified by PCR and digested with NcoI+XhoI. This fragment was cloned into the PH9L3L3 library DNA (ELN: de novo 2010 phage library SRI-021). This was transformed into *E. coli* MC1061F' cells, generating phages displaying this Fab library.

[0846] Table 24: PH9H9L1 Document Design

[0847] Location Parent AA Document 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

[0848] Table 25: PH9L3L3 Library Design

[0849]

[0850]

[0851] Biotinylated human PSMAECD was subjected to solution panning of a PSMA affinity-matured Fab-pIX library in three rounds. Phage-bound antigen was captured onto neutral avidin magnetic beads (GE Healthcare Life Science, catalog number 78152104011150) according to the manufacturer's protocol, then thoroughly washed in 1xPBST (0.05% Tween 20) and incubated for an extended period with a 500-fold molar excess of unlabeled PSMAECD from the biotinylated antigen. This panning yielded clones PSMXP46R3_59H09, PSMXP46R3_59H06, PSMXP46R3_59E03, PSMXP46R3_59C09, PSMXP46R3_59H01, PSMXP46R3_59F11, and PSMXP46R3_59F07.

[0852] To determine the expression level of the anti-PSMAfab clone, 96-well Maxisorb plates were coated overnight at 4°C with anti-human FdIgG, washed, and blocked with 3% milk-PBS-0.05% Tween for 1 hour. Phage supernatant samples were diluted 11 times in blocking buffer, serially by a 2-fold dilution, with the final wells being blank. 100 μl of these solutions were added to the coated plates for 1 hour. The plates were washed, and 100 μl of anti-F(ab')2-HRP antibody was added and incubated for 1 hour. The plates were washed and developed with 100 μl of peroxidase reagent, and the luminescence was read on an Envision microscope. Figure 31 ).

[0853] To determine the binding of the anti-PSMAfab clone to recombinant human and cynomolgus monkey proteins, 96-well Maxisorb plates were coated overnight at 4°C with 100 μl of 5 μg / ml neutral avidin. The plates were washed and blocked with 3% milk-PBS-0.05% Tween for 1 hour. Recombinant biotinylated human and cynomolgus monkey PSMA proteins were captured at 2.5 μg / ml for 1 hour at room temperature. The plates were washed and captured for 1 hour at RT with 100 μl of 2-fold serially diluted fab supernatant. The plates were washed and then incubated with 200 μl of PBST containing 0.3% milk for 2.5 hours to wash away some weakly affinistic fabs. Then, the plates were incubated with fresh 200 μl of PBST containing 0.3% milk for 30 minutes to remove even weaker affinistic fabs. The plates were washed and incubated with 100 μl of anti-F(ab')2-HRP antibody for 1 hour. Wash the plate and develop it with 100 μl of peroxidase reagent, then read the luminescence on an Envision plate. Figure 32 and Figure 33 ).

[0854] Figure 31 This indicates that protein expression in parental Fab and affinity-matured Fab is similar. The y-axis value represents the luminescence of the assay reagent, which is equal to the abundance of Fab protein on the dilution curve; higher luminescence readings indicate more protein in the wells, with protein levels decreasing after consecutive two-fold dilutions. More protein is present in the wells with affinity-matured Fab, but the increase compared to the parent is at most five-fold, as indicated by the EC50 value (which represents half the protein concentration providing the maximum response). These data suggest that... Figure 32 and Figure 33 The difference in PSMA binding characteristics is not due to the difference in Fab concentration.

[0855] Figure 32This indicates that affinity-matured fabs exhibit improved binding to the human recombinant antigen compared to parental anti-PSMA fabs (PSMB83). Similarly, the y-axis of the graph represents the luminescence value. In this case, a higher value indicates more fabs binding to the human PSMA protein. This is a measure of binding, as increased fab concentrations (along the x-axis) produce higher luminescence values. Binding of parental fabs under these conditions is negligible, as evidenced by the absence of signal even at high concentrations (hollow circles along the x-axis). Binding of affinity-matured fabs was observed at the tested concentrations, equivalent to a stronger binding affinity to the human PSMA protein. Given that the binding of parental fabs to the human PSMA protein is zero, no EC50 is generated.

[0856] Figure 33 This indicates that affinity-matured fabs exhibit improved binding to the cynomolgus recombinant antigen compared to parental anti-PSMA fabs (PSMB83). Again, the y-axis of the graph represents the luminescence value. In this case, a larger value indicates more fabs binding to the cynomolgus PSMA protein. This is a measure of binding, as increased fab concentrations (along the x-axis) produce higher luminescence values. Binding of parental fabs under these conditions is negligible, as evidenced by the absence of signal even at high concentrations (hollow circles along the x-axis). Binding of affinity-matured fabs was observed at the tested concentrations, equivalent to stronger binding to human PSMA protein. Given that binding of parental fabs to human PSMA protein is zero, no EC50 was generated for direct comparison.

[0857] Overall, compared to the parental anti-PSMAmAb (PSMB127), the phage Fab binding signature indicated improved binding to human and cynomolgus monkey recombinant antigens. This improvement was not a result of differences in Fab expression signatures, as... Figure 33 and Figure 34 As shown, the binding affinity of mature Fab to the Fab expression level is illustrated. The top five Fab candidates identified from ELISA screening were generated as monoclonal antibodies on IgG4PAA. Table 26 lists the subsequent Mab identifiers, and Tables 27 and 28 describe their variable region sequences, as well as the sequences of the heavy and light chains, respectively.

[0858] Table 26: Top five antibodies with the highest affinity for maturation identified by ELISA

[0859]

[0860] Table 27. VH and VL sequences of the top five PSMA Fab candidates

[0861]

[0862]

[0863] Table 28. Heavy and light chain sequences of the top five PSMA candidates in monoclonal antibody form on IgG4 PAA.

[0864]

[0865]

[0866]

[0867] Three different HCs and four different LCs were combined in a matrix to expand hit diversity (Table 29). Considering that methionine in CDR2 of PSMH860 is a post-translational risk, a new sequence was generated using M64L and identified as PSMH865. PSMH865 was paired with PSML160 to generate MabPSMB365.

[0868] Table 29: Matrix Form of Combinations with 3 Heavy Chains and 4 Light Chains

[0869] PSMH859 PSMH860 PSMH862 PH9L3 PSMB344 PSMB347 PSMB358 PSML158 -- PSMB361 PSMB349 PSML159 PSMB345 PSMB362 PSMB359 PSML160 PSMB346 PSMB363 PSMB360

[0870] Tables 30 and 31 show the sequences of the variable regions of the matrix recombination, as well as the sequences of the heavy and light chains, respectively.

[0871] Table 30. VH and VL sequences hit by PSMA matrix recombination

[0872]

[0873]

[0874] Table 31. Heavy and light chain sequences hit by PSMA matrix recombination.

[0875]

[0876]

[0877]

[0878]

[0879] Table 32 shows the CDR sequences for all affinity-mature PSMA hits.

[0880] Table 32. CDR sequences of PSMA hits with mature affinity

[0881]

[0882]

[0883] 3. Preparation and functional evaluation of PSMA×CD3 bispecific antibodies

[0884] 3-1 Production of PSMA×CD3 Bispecific Antibody

[0885] Two types of affinity-matured PSMA×CD3 bispecific antibodies were generated: one specific to the target arm (e.g., affinity-matured anti-PSMA), which was recombined with either the high-affinity CD3 arm, namely the CD3B376 (VHSEQ ID NO:652, VLSEQ ID NO:661; HCSEQ ID NO:640, LCSEQ ID NO:676) or the low-affinity CD3B450 (VHSEQ ID NO:657, VLSEQ ID NO:678, HCSEQ ID NO:675, LCSEQ ID NO:677) arm.

[0886] These parental mAbs are in GenMab form (Labrijn et al., 2013), with the target parent (PSMA) containing the 409R GenMab mutation (a native amino acid of IgG4), and the killing parent (CD3) containing the F405L GenMab mutation and the R409K mutation. The monospecific anti-CD3 antibody is expressed as IgG4 with Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arms) in its Fc region (numbered according to the EU index). The target parent (PSMA) is on human IgG4 with Fc substitutions S228P, F234A, and L235A. The monospecific antibody is expressed in the HEK cell line under the action of the CMV promoter.

[0887] Parental PSMA and CD3 antibodies were purified using a Protein A column with elution buffer of 100 mM NaAc pH 3.5 and neutralization buffer of 2.5 M Tris pH 7.2. The neutralized parental mAb was used to prepare a PSMA×CD3 bispecific antibody. A portion of the parental mAb was further buffer-exchanged in D-PBS (pH 7.2) buffer for analytical measurements.

[0888] After purification, controlled Fab arm exchange was performed to prepare bispecific antibodies. Parental PSMA antibody and desired parental CD3 antibody were mixed in 75 mM 2-MEA (2-mercaptoethylamine) under reducing conditions and incubated at 31°C for 4 hours or at room temperature overnight. Recombinant reactions were based on molar ratios, with a 6% excess of parental PSMA mAb used to minimize the residual parental CD3 mAb after recombination. The recombinants were then dialyzed against 1x DPBS (pH 7.2) to remove the reducing agent.

[0889] The resulting bispecific antibodies, along with the parental mAbs (i.e., PSMA, CD3, or blank) used for the recombinant reaction, are listed in Table 33.

[0890] The selected PSMA hits were also paired with the non-killing arm (blank) to generate negative controls for the testing purposes. For the control bispecific antibody B2M1, RSV antibodies in the form of IgG4PAA were generated and purified (VHSEQ ID NO: 610, VLSEQ ID NO: 611), 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.

[0891] Table 33. Bispecific antibodies against PSMA×CD3 generated from affinity-matured PSMA hits. produce

[0892]

[0893]

[0894] 3-2 Assessing the maturation of bispecific antibodies with PSMA×CD3 affinity in LNCAP cell binding

[0895] The binding of the PSMA×CD3 bispecific antibody to the PSMA-positive cell line LNCAP and the PSMA-negative cell line PC3 was tested. To assess the binding ability of the PSMA bispecific antibody, a cell binding assay (as described previously) was used. The bispecific antibody was normalized to protein concentration and then incubated with the same number of cells expressing human or cynomolgus monkey PSMA. MFI at each concentration was collected by flow cytometry and plotted as a function of concentration. Data were transformed using log10 and then plotted. Nonlinear regression of the binding curves was performed to determine the EC50.

[0896] These relative values ​​are used to rank the PSMA bound to the target cells. Figures 14 to 16 The LNCAP binding of all prepared bispecific antibodies is shown. Figure 16 None of the constructs showed binding to PSMA-negative cell lines. Figure 14 and Figure 15 In comparison with the parent Mab PS2B27, all affinity maturations showed increased binding affinity and increased cMax by shifting the curve to the left.

[0897] The interactions of affinity-matured bispecific antibodies with recombinant cynomolgus monkey PSMAECD and human PSMAECD were investigated using the ProteOnXPR36 system (BioRad) via surface plasmon resonance (SPR), as previously described for recombinant chimpanzee PSMAECD. All bispecific antibodies bound to both targets with substantially the same affinity, with KD ranging from 0.05 nM to 0.27 nM for human PSMAECD and from 0.05 nM to 0.23 nM for cynomolgus monkey PSMAECD.

[0898] 3-3 Evaluation of bispecific Abs for PSMA×CD3 affinity maturation in functional cell killing assays

[0899] 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 redirection of T cell cytotoxicity was further characterized. T-cell-mediated killing was measured using a caspase cytotoxicity assay, which indirectly measures cell killing by cleaving a fluorescent substrate with active caspase 3 / 7. Substrate cleavage produces a fluorescent DNA dye, with fluorescence confined to the cell nucleus. Throughout the assay, repeated fluorescence measurements were performed in each well using a 10X machine microscope capable of precise imaging of the wells in the same coordinate system. Target cell populations were identified based on defined size constraints and / or by the use of a secondary label. Frozen PanCD3+ T cells (purchased from BiologicalSpecialty Corporation, Colmar, PA) were isolated from normal healthy donors via negative selection. PSMA-expressing prostate cancer cells (LNCaP, C42) were cultured in RPMI1640 (purchased from Life Technologies) with a 10% HIFBS+ supplement.

[0900] Without selectivity reagents, T cells and target cells were mixed in phenol red-free RPMI with 10% FBS and supplement (Life Technologies) at a 3:1 effector-to-target ratio (E:T), and 0.6 μL of NucView caspase reagent (Essen Bioscience) was added per mL of cells according to the manufacturer's instructions. A total of 0.1 mL of cells was added to the appropriate wells of a clear 96-well flat-bottomed plate (BD Falcon). Bispecific antibodies for PS3B27 (CD3×PSMA), CD3B288 (CD3×blank), or PS3B46 (PSMA×blank) were prepared at a 2X final concentration in phenol red-free RPMI as described above, and 0.1 mL of the compound was added to each well. After incubation at room temperature for 30 minutes to minimize cell aggregation at the well edges, the plate was transferred to a Zoom Incucyte instrument (Essen Bioscience). The Incucyte instrument is located in a humidified incubator set at 37°C and 5% CO2.

[0901] The treatment definition on Incucyte was designed according to the manufacturing instructions for each cell line tested. 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. Figure 17 The data shown indicate that the curves for PS3B80, PS3B79, PS3B89, PS3B90, PS3B63, and PS3B72 shifted to the left, suggesting higher potency than PS3B27. The blank control arm did not induce cell death as expected.

[0902] Antitumor efficacy of LnCaP xenograft in tumorigenesis prevention in 3-4 humanized NSG mice

[0903] In male NOD.Cg-Prkdc cells, humanized via intraperitoneal (ip) administration of human T cells. scid Il2rg tm1WjlThe efficacy of PS3B79 and PS3B90 was evaluated in established 3DLnCaP 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 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. Tumor growth inhibition (%TGI) was calculated on day 53 post-tumor implantation (which was the last day of the study) when nine (9) animals remained in each group. Statistically significant tumor growth inhibition was observed compared to the blank × CD3 control as follows: For PS3B79 at 5 mg / kg, the observed TGI was 42% (two-way ANOVA with Bonferroni test, *p<0.0001, *p<0.0001). Figure 20 For PS3B90 at 2.5 mg / kg and 5 mg / kg, the observed TGIs were 53% and 33%, respectively (two-way ANOVA using Bonferroni test, *p<0.001). Figure 21 Therefore, CD3B376 can induce T cell activation and cytotoxicity in vivo, and leads to tumor growth inhibition in a bispecific form with high affinity for PSMA binding arms PSMB360 and PSMB365.

[0904] 4. Preparation and functional evaluation of IL1RAP×CD3 bispecific antibodies

[0905] The IL1RAP monoclonal antibodies used in and applicable to the bispecific antibodies disclosed herein are described in U.S. Patent Application Publication 20170121420A1, the entire disclosure of which is incorporated herein by reference. Fifteen monospecific IL1RAP antibodies (see Table 6 of US20170121420A1) express IgG4 with Fc substitutions for S228P, L234A, and L235A. CD3 parental mAbs express IgG4 (numbered according to EU index) with S228P, L234A, L235A, F405L, and R409K. It also produced a monospecific anti-CD3 antibody, CD3B220, which includes VH and VL regions having VH of SEQ ID NO:92 and VL of SEQ ID NO:93, and an IgG4 constant region having S228P, L234A, L235A, F405L and R409K substitutions.

[0906] The monospecific antibody was purified usi...

Claims

1. An isolated recombinant anti-CD3 antibody or an antigen-binding fragment thereof, comprising: A heavy chain and a light chain, the heavy chain comprising: a heavy chain complementary determining region (HCDR) 1 shown in the amino acid sequence of SEQ ID NO: 662, HCDR2 shown in the amino acid sequence of SEQ ID NO: 663, and HCDR3 shown in the amino acid sequence of SEQ ID NO: 664, and the light chain comprising: a light chain complementary determining region (LCDR) 1 shown in the amino acid sequence of SEQ ID NO: 671, LCDR2 shown in the amino acid sequence of SEQ ID NO: 673, and LCDR3 shown in the amino acid sequence of SEQ ID NO:

690.

2. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to claim 1, comprising: a) a heavy chain variable region comprising SEQ ID NO: 652 and a light chain variable region comprising SEQ ID NO: 661; b) a heavy chain comprising SEQ ID NO: 640 and a light chain comprising SEQ ID NO: 676; c) a heavy chain variable region comprising SEQ ID NO: 657 and a light chain variable region comprising SEQ ID NO: 678; or d) a heavy chain comprising SEQ ID NO: 675 and a light chain comprising SEQ ID NO:

677.

3. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment specifically binds to cynomolgus monkey or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of 300 nM or less.

4. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, wherein the binding affinity is 100 nM or less.

5. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, wherein the binding affinity is measured by flow cytometry or by Proteon surface plasmon resonance assay ProteOn XPR36 system at +25°C.

6. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment has one, two, three or four of the following properties: a) binds to human and cynomolgus CD3+ T lymphocytes with a calculated EC50 of 300 nM or less, and binds to HEK cells expressing cynomolgus 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 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) with an equilibrium dissociation constant (K) of 300 nM or less D ) binds to recombinant CD3d from humans (SEQ ID NO: 691), or binds to recombinant CD3e from humans (SEQ ID NO: 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 K D Measured using the Proteon surface plasmon resonance assay ProteOn XPR36 system at +25°C; c) binds to residues 1-6 of CD3 epsilon as determined by X-ray crystallography; or d) activated T cells or induced CD69 expression to a similar extent as cOKT3 or SP34-2, as determined by fluorescence activated cell sorting assay.

7. The antibody or antigen-binding fragment thereof according to claim 1, comprising at least one substitution in an antibody constant domain, the at least one substitution comprising: a) heavy chain substitutions K409R, F405L, or F405L and R409K; b) heavy chain substitutions S228P, F234A and L235A; c) heavy chain substitutions L234A, G237A, P238S, H268A, A330S and P331S, wherein the antibody is of IgG1 isotype; or d) heavy chain substitution S228P, wherein the antibody is of IgG4 isotype; The residues are numbered according to the EU index.

8. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL) of SEQ ID NO: 652 and 661, respectively.

9. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain sequence (HC) and a light chain sequence (LC) of SEQ ID NO: 640 and 676, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 1, comprising VH and VL of SEQ ID NOs: 657 and 678, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 1, comprising HC and LC of SEQ ID NOs: 675 and 677, respectively.

12. An antibody or antigen-binding fragment thereof comprising VH and VL of SEQ ID NO: 652 and 661, respectively.

13. An antibody or antigen-binding fragment thereof comprising HC and LC of SEQ ID NO: 640 and 676, respectively.

14. An antibody or antigen-binding fragment thereof comprising VH and VL of SEQ ID NOs: 657 and 678, respectively.

15. An antibody or antigen-binding fragment thereof comprising HC and LC of SEQ ID NOs: 675 and 677, respectively.

16. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is human or humanized.

17. The antibody of claim 16, wherein the antibody is of IgG4 or IgG1 isotype.

18. The antibody of claim 1, wherein the antibody is bispecific.

19. A bispecific antibody comprising a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673 and 690, respectively.

20. The bispecific antibody of claim 19, wherein the first domain and the second domain are of the IgG4 isotype, and wherein the first domain or the second domain comprises S228P, F234A, L235A, F405L and R409K heavy chain substitutions, and the other of the first domain or the second domain comprises S228P, F234A and L235A heavy chain substitutions, wherein the residues are numbered according to the EU index.

21. The bispecific antibody of claim 19, wherein the first domain and / or the second domain comprises at least one substitution in a CH3 constant domain comprising a F405L or a F405L and R409K substitution, wherein the residues are numbered according to the EU index.

22. The bispecific antibody of claim 19, wherein one of the first domain or the second domain comprises a 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.

23. The bispecific antibody of claim 19, wherein the first domain and the second domain are of the IgG4 isotype, wherein one of the first domain or the second domain comprises a S228P heavy chain substitution and the other of the first domain or the second domain comprises a S228P, F405L, and R409K heavy chain substitutions, wherein the residues are numbered according to the EU index.

24. The bispecific antibody of claim 19, wherein the first domain comprises VH and VL of SEQ ID NO: 652 and 661, respectively.

25. The bispecific antibody of claim 19, wherein the first domain comprises HC and LC of SEQ ID NO: 640 and 676, respectively.

26. The bispecific antibody of claim 19, wherein the first domain comprises VH and VL of SEQ ID NOs: 657 and 678, respectively.

27. The bispecific antibody of claim 19, wherein the first domain comprises HC and LC of SEQ ID NO: 675 and 677, respectively.

28. The bispecific antibody of claim 19, wherein the second antigen is a cell surface antigen expressed on a target cell other than an immune effector cell. The bispecific antibody of claim 28 , wherein the cell surface antigen is a tumor-associated antigen.

30. The bispecific antibody of claim 19, wherein the second antigen is CD33, IL1RAP, PSMA or TMEFF2.

31. A pharmaceutical composition comprising the antibody according to any one of the preceding claims and a pharmaceutically acceptable carrier.

32. A polynucleotide encoding the antibody according to any one of claims 1 to 30.

33. A vector comprising the polynucleotide according to claim 32.

34. A host cell comprising the vector according to claim 33.

35. A method for preparing an antibody according to any one of claims 1 to 30, comprising: Cultivating the host cell according to claim 34 under conditions allowing the expression of the antibody, and recovering the antibody produced by the host cell.

36. Use of the bispecific antibody according to any one of claims 18 to 30 in the preparation of a medicament for treating cancer in a subject, wherein the second antigen is a tumor-associated antigen.

37. The use according to claim 36, wherein the cancer is a solid tumor or a hematological malignancy.

38. The use according to claim 37, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, pancreatic cancer, renal cancer or urothelial carcinoma.

39. The use according to claim 37, wherein the solid tumor is renal clear cell carcinoma.

40. The use according to claim 37, wherein the solid tumor is metastatic adenocarcinoma of the liver.

41. The use according to claim 38, wherein the prostate cancer is refractory prostate cancer, prostatic intraepithelial neoplasia or androgen-independent prostate cancer.

42. The use according to claim 38, wherein the prostate cancer is malignant prostate cancer.

43. The use according to claim 37, 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).

44. The antibody of any one of claims 1 to 30 for use in therapy.

45. The use of claim 36, wherein the medicament further comprises a second therapeutic agent.

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