Anti-CD3 antibodies and uses thereof

Abstract

To provide bispecific antibodies that recruit cytolytic T cells to kill tumor cells with reduced toxicity and favorable manufacturing profiles.SOLUTION: The invention relates to an antibody that specifically binds to CD3. The invention relates to an antibody that specifically binds to PSMA. The invention relates to an antibody that specifically binds to CD3 and PSMA. The invention relates to an antibody that specifically binds to IL1RAP. The invention relates to an antibody that specifically binds to CD33. The invention relates to an antibody that specifically binds to CD3 and IL1RAP. The invention relates to an antibody that specifically binds to CD3 and CD33. The invention relates to an antibody that specifically binds to TMEFF2. The invention relates to an antibody that specifically binds to CD3 and TMEFF2 The invention relates to the fragments of the antibodies, polynucleotides encoding the antibodies or fragments thereof, and methods of making and using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Sequence Listing) This application has been filed electronically in ASCII format and is incorporated by reference in its entirety. The above ASC created on May 3, 2019, includes a sequence listing incorporated herein. II copy JBI5135WOPCT1_Sequence_listing.tx The file name is t and is 588KB in size.

[0002] FIELD OF THE INVENTION The disclosure provided herein provides antibodies capable of specifically binding to human and non-human CD3. Anti-cluster of differentiation 3 (CD3) antibodies and antigen-binding fragments thereof, particularly those derived from non-human mammals (e.g., Anti-CD3 antibodies and antigen-binding fragments that are cross-reactive with CD3 of humans (e.g., cynomolgus monkeys); and a PSMA antibody capable of specifically binding to non-human prostate-specific membrane antigen (PSMA). and antigen-binding fragments thereof; capable of specifically binding to human and non-human IL1RAP IL1RAP antibodies and antigen-binding fragments thereof; specifically bind to human and non-human CD33 CD33 antibodies and antigen-binding fragments thereof capable of inhibiting the activity of CD3, PSMA, and IL1RAP , CD33, CD3 and PSMA, CD3 and IL1RAP, or CD3 and CD33 The present disclosure also relates to bispecific antibodies capable of specifically binding to cancer, autoimmune diseases, and the like. and / or the use of such antibodies and antigen-binding fragments in the treatment of inflammatory diseases and other conditions. It also relates to the use of the pieces. [Background technology]

[0003] Bispecific antibodies and antibody fragments recruit cytolytic T cells to kill tumor cells. However, many of the T cell-engaging bispecific antibodies have not been clinically investigated. Its use is hampered by challenges including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing issues. Therefore, tumor cell lines exhibiting low toxicity and favorable manufacturing profiles are being considered. There is a great need for bispecific antibodies that recruit cytolytic T cells to kill cells. do.

[0004] The human CD3 T cell antigen receptor protein complex consists of six distinct chains: the CD3γ chain (S wissProt P09693), CD3δ chain (SwissProt P04234) , two CD3ε chains (SwissProt P07766), and one CD3ζ chain homolog It consists of a dimer (SwissProt P20963) (ε γ:ε δ:ζζ) It associates with the α and β chains of the T cell receptor. This complex mediates the expression of several intracellular signals. The CD3 complex plays an important role in linking antigen recognition to the signal transduction pathway. CD3 mediates signaling, resulting in T cell activation and proliferation. CD3 is essential for the immune response. be. Summary of the Invention [Means for solving the problem]

[0005] Heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 662, HCDR comprising SEQ ID NO: 663 a heavy chain comprising an HCDR2 and an HCDR3 comprising SEQ ID NO: 664, and SEQ ID NO: 671 Light chain complementarity determining region (LCDR) 1, LCDR2 comprising SEQ ID NO: 673, and SEQ ID NO: 6 a light chain comprising an LCDR3 comprising SEQ ID NO: 652; a heavy chain variable region comprising SEQ ID NO: 66 a heavy chain comprising SEQ ID NO: 640 and a light chain comprising SEQ ID NO: 676; or an HCDR1 comprising SEQ ID NO: 662, an HCDR2 comprising SEQ ID NO: 663, and the sequence a heavy chain comprising an HCDR3 comprising SEQ ID NO: 664, and an LCDR1 comprising SEQ ID NO: 773, a light chain comprising an LCDR2 comprising SEQ ID NO: 673 and an LCDR3 comprising SEQ ID NO: 690; a heavy chain variable region comprising SEQ ID NO: 657 and a light chain variable region comprising SEQ ID NO: 678; or SEQ ID NO: 675 and a light chain comprising SEQ ID NO: 678, or an isolated recombinant anti-CD3 antibody or Antigen-binding fragments of are provided herein.

[0006] In addition, it was detected in cynomolgus monkeys (Macaca fascicularis) or humans with a binding affinity of approximately 300 nM or less. Isolated recombinant human CD3d, or CD3e, or CD3e and CD3d Anti-CD3 antibodies or antigen-binding fragments thereof are also provided herein.

[0007] In some embodiments, the isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof has the following characteristics: Having one, two, three, or four of the following sexes: Binds to human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 20 nM or less , binds to cynomolgus monkey CD3-expressing HEK cells with a calculated EC50 of 40 nM or less, and CD3+ Calculated EC50 between binding to T lymphocytes and binding to cynomolgus monkey CD3-expressing HEK cells If the difference is less than 5-fold, calculate the EC50 using flow cytometry for whole cell measured in binding assays; Human recombinant CD3d (SEQ ID NO: 691) with an equilibrium dissociation constant (KD) of 12 nM or less ), or recombinant CD3e of human origin (SEQ ID NO: 636) or recombinant CD3e of cynomolgus monkey origin of recombinant CD3d (SEQ ID NO: 692) and the KD was Measured using the plasmon resonance assay ProteOn XPR36 system; binds to residues 1-6 of CD3e as determined by X-ray crystallography; or cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay To a similar extent, it activates T cells or induces CD69 expression.

[0008] In some embodiments, the antibodies or antigen-binding fragments thereof described herein each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 671, 673, and 690 , HCDR3, LCDR1, LCDR2, and LCDR3; SEQ ID NOs: 652 and 653, respectively. 661; or the HC and LC of SEQ ID NOs: 640 and 676, respectively.

[0009] In some embodiments, the antibodies or antigen-binding fragments thereof described herein each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 773, 673, and 690 , HCDR3, LCDR1, LCDR2, and LCDR3; SEQ ID NOs: 657 and 658, respectively. 678; or the HC and LC of SEQ ID NOs: 675 and 677, respectively.

[0010] Furthermore, the antibody comprises a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. a bispecific antibody comprising two domains, the first domain being an antibody described herein;

[0013] Provided herein are bispecific antibodies comprising the antibody or antigen-binding fragment thereof.

[0011] The bispecific antibodies described herein are represented by SEQ ID NOs: 662, 663, 664, and 67, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR of 1, 673, and 670 2, and the first domain containing LCDR3, and SEQ ID NOs: 697, 683, 698, respectively , 699, 792, and 686 HCDR1, HCDR2, HCDR3, LCDR1, L a second domain comprising CDR2, and LCDR3;

[0012] The bispecific antibodies described herein comprise the VH and VH sequences of SEQ ID NOs: 652 and 661, respectively. a first domain comprising VH and VL of SEQ ID NOs: 681 and 682, respectively; and two domains.

[0013] The bispecific antibodies described herein comprise the HC and L of SEQ ID NOs: 640 and 676, respectively. The first domain includes C, and the second domain includes HC and LC of SEQ ID NOs: 679 and 680, respectively. and two domains.

[0014] The bispecific antibodies described herein are represented by SEQ ID NOs: 662, 663, 664, and 67, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR of 1, 673, and 670 2, and the first domain comprising LCDR3, and SEQ ID NOs: 704, 705, and 706, respectively , 707, 708, and 709 of HCDR1, HCDR2, HCDR3, LCDR1, L and a second domain comprising CDR2, and LCDR3.

[0015] The bispecific antibodies described herein comprise the VH and VH sequences of SEQ ID NOs: 652 and 661, respectively. a first domain comprising VH and VL of SEQ ID NOs: 702 and 703, respectively; and two domains.

[0016] The bispecific antibodies described herein comprise the HC and L of SEQ ID NOs: 640 and 676, respectively. The first domain comprises C, and the second domain comprises HC and LC of SEQ ID NOs: 700 and 701, respectively. and two domains.

[0017] The present disclosure further provides recombinant cynomolgus or human CD3d or a first domain that binds to cells expressing CD3e, and the binding to the cells is detected by flow cytometry; The first domain, measured by immunohistochemistry, and the second domain, which specifically binds to PSMA. and an isolated bispecific CD3xPSMA antibody comprising:

[0018] In some embodiments, the bispecific CD3xPSMA antibody comprises a first domain Including: Binds to human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 20 nM or less , binds to cynomolgus monkey CD3-expressing HEK cells with a calculated EC50 of 40 nM or less, and CD3+ Calculated EC50 between binding to T lymphocytes and binding to cynomolgus monkey CD3-expressing HEK cells If the difference is less than 5-fold, calculate the EC50 using flow cytometry for whole cell measured in binding assays; Human recombinant CD3d (SEQ ID NO: 691) with an equilibrium dissociation constant (KD) of 12 nM or less ), or binds to recombinant CD3e of human origin (SEQ ID NO: 636), or binds to recombinant CD3e of cynomolgus monkey origin Binds to recombinant CD3d (SEQ ID NO: 692) derived from cynomolgus monkeys, or recombinant CD3 e (SEQ ID NO: 693) and the KD was measured by Proteon surface plasmon resonance at +25°C. Assay measured using the ProteOn XPR36 system; The oxidation of methionine and tryptophan was also detected by peptide mapping analysis. It does not show oxidation, deamidation of asparagine, or isomerization of asparagine. Not shown; binds to residues 1-6 of CD3e as determined by X-ray crystallography; or cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay To a similar extent, it activates T cells or induces CD69 expression.

[0019] Also described are pharmaceutical compositions comprising an antibody described herein and a pharmaceutically acceptable carrier. will be done.

[0020] The present disclosure further provides a method for producing an antibody or antigen-binding fragment thereof described herein under conditions in which the antibody is expressed. culturing a host cell containing a vector containing a polynucleotide encoding said and recovering the antibody produced by the host cell. The method for producing a bispecific CD3xPSMA antibody comprises the steps of: A monospecific bivalent CD3 antibody with an HC1 and two identical LC1s is A monospecific bivalent PSMA antibody with a C2 and two identical LC2s at a molar ratio of approximately 1:1 introducing a reducing agent into the mixture; and heating the mixture to about 90°C. Incubating for 1 minute to about 6 hours, removing the reducing agent, and Purifying a bispecific CD3xPSMA antibody containing the LC1, the HC2, and the LC2 and

[0021] A method of treating cancer in a subject, comprising administering a therapeutically effective amount of steroids to a subject for a period of time sufficient to treat the cancer. administering a therapeutically effective amount of the isolated antibody to said subject in need thereof. is further described.

[0022] The present disclosure also provides kits comprising the antibodies described herein. It may further comprise reagents and instructions for use for detection. [Brief explanation of the drawings]

[0023] The Summary of the Invention, as well as the Detailed Description below, should be read in conjunction with the accompanying drawings. For purposes of illustrating the disclosed antibodies and methods, examples of such antibodies and methods are provided. Exemplary embodiments are shown in the drawings, although the antibodies and methods may be modified to suit the specific embodiments disclosed. The drawings are as follows: [Figure 1A] Figure 1 shows anti-CD3 antibodies produced in OmniRat. The VH (Figure 1A) and VL (Figure 1B) sequences of active anti-CD3 mAbs produced in OmniRat were aligned with human germline sequences from IMGT. CDR regions are underlined. Sequence differences are indicated in bold. Figure 1A discloses SEQ ID NOS: 651, 651, 653, 656, 655, 20, 654, and 717, and Figure 1B discloses SEQ ID NOS: 658, 688, 660, 659, 659, 659, 659, and 718, all in order of appearance, respectively. [Figure 1B] Figure 1 shows anti-CD3 antibodies produced in OmniRat. The VH (Figure 1A) and VL (Figure 1B) sequences of active anti-CD3 mAbs produced in OmniRat were aligned with human germline sequences from IMGT. CDR regions are underlined. Sequence differences are indicated in bold. Figure 1A discloses SEQ ID NOS: 651, 651, 653, 656, 655, 20, 654, and 717, and Figure 1B discloses SEQ ID NOS: 658, 688, 660, 659, 659, 659, 659, and 718, all in order of appearance, respectively. [Figure 2] 1 shows a cell-based binding assay to assess the binding capacity of individual rat hybridoma supernatants to human purified CD3+ T lymphocytes. [Figure 3]1 shows a cell-based binding assay to assess the binding capacity of individual rat hybridoma supernatants to cynomolgus monkey purified CD3+ T lymphocytes. [Figure 4] 1 shows the results of a competition assay in which hybridoma supernatants were evaluated for their ability to compete with the commercially available anti-human CD3 antibody SP34-2, which has a known epitope and is cross-reactive with cynomolgus monkey CD3. [Figure 5] Representative binding curves of anti-CD3 antibodies on primary human T cells are shown. [Figure 6] Representative competitive binding curves of anti-CD3 antibodies with AlexaFluor488-conjugated SP34-2 on primary human T cells are shown. [Figure 7] Figure 1 shows binding of light chain (LC) engineered BLW-2E6 mAb to primary human T cells. [Figure 8] Figure 1 shows binding of heavy chain (HC) engineered BLW-2E6 to primary human T cells. [Figure 9] Figure 1 shows AlexaFluor™ (AF) 488 CD3 saturation in human T cells by FACS analysis. The resulting mean fluorescence intensity values ​​were plotted as a function of antibody molecule concentration. Kd values ​​were derived for each donor, and the average value was obtained. The saturation binding constant (KdT) for human T cells was derived to be 5.6±1.0 nM (n=4) and used herein to determine Kd binding affinity. One human donor was excluded due to failure to meet the criterion of at least 60% viability during analysis. The "LS" identifier in the figure legend refers to the individual human T cell donor. [Figure 10] Figure 1 shows the inhibition curves of bivalent anti-CD3 antibodies, CD3B376 and CD3B450, for competition for binding to AlexaFluor488 SP-34 anti-CD3 antibody. IC50 values ​​were derived to be 29 and 60 nM, respectively. [Figure 11] 1 shows a sensorgram of BLW-2E6 mutants binding to hCD3ε(1-27)-Tn25. [Figure 12A]The thermal stability of anti-CD3 antibodies CD3B376 (Figure 12A), CD3B450 (Figure 12B), CD3B389 (Figure 12C), and CD3B467 (Figure 12D) by DSC is shown, as well as an overlay of the thermograms of all candidates (Figure 12E). [Figure 12B] The thermal stability of anti-CD3 antibodies CD3B376 (Figure 12A), CD3B450 (Figure 12B), CD3B389 (Figure 12C), and CD3B467 (Figure 12D) by DSC is shown, as well as an overlay of the thermograms of all candidates (Figure 12E). [Figure 12C] The thermal stability of anti-CD3 antibodies CD3B376 (Figure 12A), CD3B450 (Figure 12B), CD3B389 (Figure 12C), and CD3B467 (Figure 12D) by DSC is shown, as well as an overlay of the thermograms of all candidates (Figure 12E). [Figure 12D] The thermal stability of anti-CD3 antibodies CD3B376 (Figure 12A), CD3B450 (Figure 12B), CD3B389 (Figure 12C), and CD3B467 (Figure 12D) by DSC is shown, as well as an overlay of the thermograms of all candidates (Figure 12E). [Figure 12E] The thermal stability of anti-CD3 antibodies CD3B376 (Figure 12A), CD3B450 (Figure 12B), CD3B389 (Figure 12C), and CD3B467 (Figure 12D) by DSC is shown, as well as an overlay of the thermograms of all candidates (Figure 12E). [Figure 13A] A comparison of the thermal stability of anti-CD3 antibodies is shown; overlay of thermograms of CD3B376 and CD3B389 (Figure 13A); overlay of thermograms of CD3B450 and CD3B467 (Figure 13B). [Figure 13B] A comparison of the thermal stability of anti-CD3 antibodies is shown; overlay of thermograms of CD3B376 and CD3B389 (Figure 13A); overlay of thermograms of CD3B450 and CD3B467 (Figure 13B). [Figure 14] 1 shows LNCAP cell binding of a subset of affinity-matured PSMAxCD3 bispecific antibodies. [Figure 15] 1 shows LNCAP cell binding of a subset of affinity-matured PSMAxCD3 bispecific antibodies. [Figure 16] 1 shows PSMA-negative PC3 cell binding of affinity-matured PSMAxCD3 bispecific antibodies. [Figure 17] 1 shows the results of PSMAxCD3 affinity matured bispecific Ab in a functional cell killing assay. [Figure 19] Antibody-antigen interactions in the CD3B334:CD3 complex are shown, with CD3 residues in ovals and CD3B334 residues in boxes. [Figure 20] 1 shows primary human and cynomolgus monkey T cell-based assays used to determine the ability of hybridoma hits to activate T cells as measured by CD69 activation. [Figure 21] Figure 1 shows the antitumor efficacy of PS3B79 in LnCAP AR.TB human prostate xenografts in T cell-humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and the results are presented as the mean tumor volume, expressed in mm ± SEM (*, p<0.0001). [Figure 22] Figure 1 shows the antitumor efficacy of PS3B90 in LnCAP AR.TB human prostate xenografts in T cell-humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and the results are presented as the mean tumor volume, expressed in mm ± SEM (*, p<0.001). [Figure 23A] Figure 23 shows titration curves of anti-PSMA phage panning hits against human LNCaP cells. Figure 23A shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 23B shows M52 and M110; Figure 23C shows M85, M87, and M81; and Figure 23D shows M52 and M84. In Figure 23D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCaP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 23B] Figure 23 shows titration curves of anti-PSMA phage panning hits against human LNCaP cells. Figure 23A shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 23B shows M52 and M110; Figure 23C shows M85, M87, and M81; and Figure 23D shows M52 and M84. In Figure 23D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCaP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 23C] Figure 23 shows titration curves of anti-PSMA phage panning hits against human LNCaP cells. Figure 23A shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 23B shows M52 and M110; Figure 23C shows M85, M87, and M81; and Figure 23D shows M52 and M84. In Figure 23D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCaP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 23D]Figure 23 shows titration curves of anti-PSMA phage panning hits against human LNCaP cells. Figure 23A shows the titration curves of hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 23B shows M52 and M110; Figure 23C shows M85, M87, and M81; and Figure 23D shows M52 and M84. In Figure 23D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCaP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 24A] Figure 24 shows titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA HEK). Figure 24A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 24B shows M52 and M110; Figure 24C shows M81, M52, M85, and M87; and Figure 24D shows M52 and M84. In Figure 24D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 24B]Figure 24 shows titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA HEK). Figure 24A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 24B shows M52 and M110; Figure 24C shows M81, M52, M85, and M87; and Figure 24D shows M52 and M84. In Figure 24D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 24C] Figure 24 shows titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA HEK). Figure 24A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 24B shows M52 and M110; Figure 24C shows M81, M52, M85, and M87; and Figure 24D shows M52 and M84. In Figure 24D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 24D]Figure 24 shows titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against chimpanzee-PSMA HEK). Figure 24A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 24B shows M52 and M110; Figure 24C shows M81, M52, M85, and M87; and Figure 24D shows M52 and M84. In Figure 24D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMG5 (cynomolgus-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 25A] Figure 25 shows titration curves of anti-PSMA phage panning hits against cynomolgus monkey PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against cynomolgus monkey-PSMA HEK). Figure 25A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 25B shows M52 and M110; Figure 25C shows M81, M52, M85, and M87; and Figure 25D shows M52 and M84. In Figure 25D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMGS (cynomolgus monkey-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 25B]Figure 25 shows titration curves of anti-PSMA phage panning hits against cynomolgus monkey PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against cynomolgus monkey-PSMA HEK). Figure 25A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 25B shows M52 and M110; Figure 25C shows M81, M52, M85, and M87; and Figure 25D shows M52 and M84. In Figure 25D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMGS (cynomolgus monkey-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 25C] Figure 25 shows titration curves of anti-PSMA phage panning hits against cynomolgus monkey PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against cynomolgus monkey-PSMA HEK). Figure 25A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 25B shows M52 and M110; Figure 25C shows M81, M52, M85, and M87; and Figure 25D shows M52 and M84. In Figure 25D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMGS (cynomolgus monkey-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 25D]Figure 25 shows titration curves of anti-PSMA phage panning hits against cynomolgus monkey PSMA-expressing HEK cells. (Mammalian Fab supernatant titration curves of anti-PSMA phage panning hits against cynomolgus monkey-PSMA HEK). Figure 25A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; Figure 25B shows M52 and M110; Figure 25C shows M81, M52, M85, and M87; and Figure 25D shows M52 and M84. In Figure 25D, mammalian-expressed supernatants were normalized for Fab expression via octet and titrated against either human LNCAP, PSMGS (cynomolgus monkey-PSMA HEK), or PSMG9 (chimpanzee-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted against Fab concentration using GraphPad Prizm. [Figure 26] The overall structure of PSMM84 Fab bound to human PSMA ECD homodimer is shown. [Figure 27] A proximity view of the major interactions of PSMA with the PSMB83 light chain is shown. [Figure 28] A close-up view of the major interactions of PSMA with the PSMB83 heavy chain is shown. [Figure 29] 1 shows a comparison of the epitope residues of PSMB83 within the sequences of human (SEQ ID NO: 719), mouse (SEQ ID NO: 720), and cynomolgus monkey (cyno) (SEQ ID NO: 721) PSMA. Epitope residues are shaded, and sequence differences are underlined. [Figure 30] The paratope residues of PSMB83 are shown. CDRs are underlined and paratope residues are shaded. Figure 30 discloses SEQ ID NOS: 722 to 727 in the order of appearance. [Figure 31] An interaction map including the direct contacts formed between PSMA and PSMM84 is shown, with van der Waals interactions indicated by dashed lines, solid lines being hydrogen bonds, and arrows pointing to backbone atoms. [Figure 32]Expression levels of anti-PSMA Fab clones derived from PSMM84 compared to that of parental PSMB83 are shown. Raw luminescence counts were plotted against log concentration. [Figure 33] Figure 1 shows the binding of anti-PSMA Fab clones derived from PSMB83 to human PSMA compared to the binding of the parent PSMM84. Raw luminescence counts were plotted against log concentration. [Figure 34] Figure 1 shows the binding of PSMB83-derived anti-PSMA Fab clones to cynomolgus monkey PSMA compared to the binding of parental PSMB83. Raw luminescence counts were plotted against log concentration. [Figure 35] Figure 1 shows IC3B19 and IC3B34 ex vivo mediated T cell cytotoxicity of LAMA-84 20 cells in whole blood after 48 hours. The concentrations of IC3B19 and IC3B34 are provided in the table below the figure. [Figure 36] Figure 1 shows IC3B19 and IC3B34 ex vivo mediated T cell activation in whole blood after 48 hours. The concentrations of IC3B19 and IC3B34 are provided in the table below the figure. [Figure 37A] Figure 37 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-1β release after 24 hours (Figure 37A) and corresponding 4PL regression parameter estimates (Figure 37B) are shown. [Figure 37B]Figure 37 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-1β release after 24 hours (Figure 37A) and corresponding 4PL regression parameter estimates (Figure 37B) are shown. [Figure 38A] Figure 38 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-1β release after 48 hours (Figure 38A) and corresponding 4PL regression parameter estimates (Figure 38B) are shown. [Figure 38B] Figure 38 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-1β release after 48 hours (Figure 38A) and corresponding 4PL regression parameter estimates (Figure 38B) are shown. [Figure 39A]Figure 39 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-2 release after 24 hours (Figure 39A) and corresponding 4PL regression parameter estimates (Figure 39B) are shown. [Figure 39B] Figure 39 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-2 release after 24 hours (Figure 39A) and corresponding 4PL regression parameter estimates (Figure 39B) are shown. [Figure 40A] Figure 40 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-2 release after 48 hours (Figure 40A) and corresponding 4PL regression parameter estimates (Figure 40B) are shown. [Figure 40B]Figure 40 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-2 release after 48 hours (Figure 40A) and corresponding 4PL regression parameter estimates (Figure 40B) are shown. [Figure 41A] Figure 41 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-4 release after 24 hours (Figure 41A) and corresponding 4PL regression parameter estimates (Figure 41B) are shown. [Figure 41B] Figure 41 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-4 release after 24 hours (Figure 41A) and corresponding 4PL regression parameter estimates (Figure 41B) are shown. [Figure 42]IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-4 release after 48 hours is shown. [Figure 43A] Figure 43 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-6 release after 24 hours (Figure 43A) and corresponding 4PL regression parameter estimates (Figure 43B) are shown. [Figure 43B] Figure 43 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-6 release after 24 hours (Figure 43A) and corresponding 4PL regression parameter estimates (Figure 43B) are shown. [Figure 44]IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-6 release after 48 hours is shown. [Figure 45] IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-8 release after 24 hours is shown. [Figure 46] IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-8 release after 48 hours is shown. [Figure 47A] Figure 47 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-10 release after 24 hours (Figure 47A) and corresponding 4PL regression parameter estimates (Figure 47B) are shown. [Figure 47B]Figure 47 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-10 release after 24 hours (Figure 47A) and corresponding 4PL regression parameter estimates (Figure 47B) are shown. [Figure 48A] Figure 48 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-10 release after 48 hours (Figure 48A) and corresponding 4PL regression parameter estimates (Figure 48B) are shown. [Figure 48B] Figure 48 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-10 release after 48 hours (Figure 48A) and corresponding 4PL regression parameter estimates (Figure 48B) are shown. [Figure 49]IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-12p70 release after 24 hours is shown. [Figure 50] IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-12p70 release after 48 hours is shown. [Figure 51] IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells) is shown. Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-13 release after 24 hours is shown. [Figure 52A] Figure 52 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-13 release after 48 hours (Figure 52A) and corresponding 4PL regression parameter estimates (Figure 52B) are shown. [Figure 52B] Figure 52 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IL-13 release after 48 hours (Figure 52A) and corresponding 4PL regression parameter estimates (Figure 52B) are shown. [Figure 53A] Figure 25 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IFN-γ release after 24 hours (Figure 25 53A) and corresponding 4PL regression parameter estimates (Figure 53B) are shown. [Figure 53B] Figure 25 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IFN-γ release after 24 hours (Figure 25 53A) and corresponding 4PL regression parameter estimates (Figure 53B) are shown. [Figure 54A]Figure 54 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IFN-γ release after 48 hours (Figure 54A) and corresponding 4PL regression parameter estimates (Figure 54B) are shown. [Figure 54B] Figure 54 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell IFN-γ release after 48 hours (Figure 54A) and corresponding 4PL regression parameter estimates (Figure 54B) are shown. [Figure 55A] Figure 55 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell TNF-α release after 24 hours (Figure 55A) and corresponding 4PL regression parameter estimates (Figure 55B) are shown. [Figure 55B]Figure 55 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell TNF-α release after 24 hours (Figure 55A) and corresponding 4PL regression parameter estimates (Figure 55B) are shown. [Figure 56A] Figure 56 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell TNF-α release after 48 hours (Figure 56A) and corresponding 4PL regression parameter estimates (Figure 56B) are shown. [Figure 56B] Figure 56 shows IC3B19- and IC3B34-mediated association of T cells and the IL1RAP+ target cell line LAMA-84 (endogenously and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T cell activation assays spiked with exogenous LAMA-84 IL1RAP+ tumor cell lines. Statistically significant differences are indicated in bold text. IC3B19- and IC3B34-mediated T cell TNF-α release after 48 hours (Figure 56A) and corresponding 4PL regression parameter estimates (Figure 56B) are shown. [Figure 57] We show that IC3B19 and IC3B34, but not bispecific antibodies with null arms (IAPB57 x B23B49 or B23B39 x CD3B219), induced target-specific cytotoxicity in NCI-H1975 cells. In this assay, the cytotoxicity EC5O differs by 3-fold between IC3B19 and IC3B34, with values ​​of 0.018 and 0.057 nM, respectively. [Figure 58] Figure 1 shows the antitumor efficacy of IAPB57xCD3B376 in H1975 human NSCLC xenografts in T cell-humanized NSG mice. Subcutaneous H1975 tumors were measured twice weekly and the results are presented as the mean tumor volume, expressed in mm ± SEM, *p<0.0001. [Figure 59] A comparison of the isoelectric points of various anti-PSMA constructs is shown. [Figure 60] The wavelength shift is plotted relative to the CNT05825 control molecule. The control CNT0607 exhibits characteristically strong self-interactions. Error bars represent the standard deviation from triplicate measurements. [Figure 61] 1 shows a comparison of retention times of IgG and control columns in an anti-PSMA cross-interaction assay. [Figure 62A] Figure 2 shows ex vivo evaluation of CD33xCD3 bispecific antibodies using anti-CD3 arms CD3B219 and CD3B376 for blast cytotoxicity and T cell activation in fresh AML patient whole blood. Figure 2A shows the percent of total cytotoxicity of AML cells using CD33 bispecific antibodies or a CD3x null control. Figure 2B shows T cell activation induced by CD33 bispecific antibodies or a CD3x null control. No Fc blocker was added. [Figure 62B] Figure 2 shows ex vivo evaluation of CD33xCD3 bispecific antibodies using anti-CD3 arms CD3B219 and CD3B376 for blast cytotoxicity and T cell activation in fresh AML patient whole blood. Figure 2A shows the percent of total cytotoxicity of AML cells using CD33 bispecific antibodies or a CD3x null control. Figure 2B shows T cell activation induced by CD33 bispecific antibodies or a CD3x null control. No Fc blocker was added. [Figure 63A]Figure 3 shows a CD33xCD3 T cell-mediated cytotoxicity assay. A CD33xCD3 bispecific antibody using anti-CD3 arms CD3B219 and anti-CD3B376 was incubated with human pan-T cells and AML cell lines that were either wild-type (KG1, Figure 3A), heterozygous (SH2, Figure 3B), or homozygous (OCIAML3, Figure 3C) for the CD33 SNP rs12459419 mutation. After 48 hours at 37°C and 5% CO2, total tumor cell cytotoxicity was measured by flow cytometry. [Figure 63B] Figure 3 shows a CD33xCD3 T cell-mediated cytotoxicity assay. A CD33xCD3 bispecific antibody using anti-CD3 arms CD3B219 and anti-CD3B376 was incubated with human pan-T cells and AML cell lines that were either wild-type (KG1, Figure 3A), heterozygous (SH2, Figure 3B), or homozygous (OCIAML3, Figure 3C) for the CD33 SNP rs12459419 mutation. After 48 hours at 37°C and 5% CO2, total tumor cell cytotoxicity was measured by flow cytometry. [Figure 63C] Figure 3 shows a CD33xCD3 T cell-mediated cytotoxicity assay. A CD33xCD3 bispecific antibody using anti-CD3 arms CD3B219 and anti-CD3B376 was incubated with human pan-T cells and AML cell lines that were either wild-type (KG1, Figure 3A), heterozygous (SH2, Figure 3B), or homozygous (OCIAML3, Figure 3C) for the CD33 SNP rs12459419 mutation. After 48 hours at 37°C and 5% CO2, total tumor cell cytotoxicity was measured by flow cytometry. [Figure 64A] Ex vivo evaluation of C33B904 antibody paired with either CD3B219 or CD3B376 on cytotoxicity of MOLM-13 cells exogenously added to normal healthy human whole blood (N=6 donors): Percent cytotoxicity of MOLM-13 cells (FIG. 4A) and CD33+CD14+ monocytes (FIG. 4B) using CD33xCD3 bispecific antibodies and respective null x CD3 controls at 48 hours is shown. [Figure 64B]Ex vivo evaluation of C33B904 antibody paired with either CD3B219 or CD3B376 on cytotoxicity of MOLM-13 cells exogenously added to normal healthy human whole blood (N=6 donors): Percent cytotoxicity of MOLM-13 cells (FIG. 4A) and CD33+CD14+ monocytes (FIG. 4B) using CD33xCD3 bispecific antibodies and respective null x CD3 controls at 48 hours is shown. [Figure 65A] Figure 5 shows ex vivo evaluation of CD33xCD3 bispecific antibodies using anti-CD3 arms CD3B219 and CD3B376 on monocyte cytotoxicity and T cell activation in fresh whole blood from six normal cynomolgus donors. Figure 5A shows the percent of total cytotoxicity of CD33+CD14+ cynomolgus monocytes using the CD33 bispecific antibody or the CD3x null control. Figure 5B shows T cell activation induced by the CD33 bispecific antibody or its CD3x null control. No Fc blocking agent was added. [Figure 65B] Figure 5 shows ex vivo evaluation of CD33xCD3 bispecific antibodies using anti-CD3 arms CD3B219 and CD3B376 on monocyte cytotoxicity and T cell activation in fresh whole blood from six normal cynomolgus donors. Figure 5A shows the percent of total cytotoxicity of CD33+CD14+ cynomolgus monocytes using the CD33 bispecific antibody or the CD3x null control. Figure 5B shows T cell activation induced by the CD33 bispecific antibody or its CD3x null control. No Fc blocking agent was added. [Figure 66] Figure 1 shows the antitumor efficacy of C3CB189 in MOLM-13 human AML xenografts in T cell-humanized NSG mice. Disseminated MOLM-13 tumors were imaged twice weekly for bioluminescence (BLI), and the results are presented as mean radiance (p / s / cm² / sr) ± SEM (n=8-10 / group). *p≤0.0001 for treated vs. control, calculated by two-way ANOVA with Bonferroni test. [Figure 67]Survival of animals treated with C3CB189 in MOLM-13 human AML xenografts in T cell-humanized NSG mice is shown. Survival of MOLM-13-bearing mice is graphically represented using Kaplan-Meier curves and assessed by the log-rank (Mantel-Cox) test. *p ≤ 0.0001 for treated vs. control groups. [Figure 68] Antitumor efficacy of C3CB88 in MOLM-13 human AML xenografts in T cell-humanized NSG mice. Disseminated MOLM-13 tumors were imaged twice weekly for bioluminescence (BLI), and results are presented as mean radiance (p / s / cm² / sr) ± SEM (n=8-10 / group). *p≦0.0001 for treated vs. control, calculated by two-way ANOVA with Bonferroni test. [Figure 69] Survival of animals treated with C3CB88 in MOLM-13 human AML xenografts in T cell-humanized NSG mice is shown. Survival of MOLM-13-bearing mice is graphically represented using Kaplan-Meier curves and assessed by the log-rank (Mantel-Cox) test. *p ≤ 0.05 for treated vs. control groups. [Figure 70]

[0023] Figure 1 shows an alignment of selected anti-TMEFF2 antibody heavy chain variable regions (VH). VH regions are identified by the SEQ ID NO at the beginning of each line. [Figure 71]

[0023] Figure 1 shows an alignment of select anti-TMEFF2 antibody light chain variable regions (VL). VH regions are identified by the SEQ ID NO at the beginning of each line. [Figure 72] 1 shows the reduction in mean tumor volume for each mouse treated with 0.5 mg / kg TMCB132 in an ex vivo LnCaP prostate cancer model in male NGS mice. [Figure 73] Shows the efficacy of TMEB762xCD3B376 in established LNCaP xenografts in T cell-humanized NSG mice. [Figure 74] 1 shows T cell activation in LnCaP prostate cancer cells in response to administration of TMCB132. [Figure 75] 1 shows T cell-mediated cytotoxicity of TMCB132. [Figure 76] 1 shows the antitumor efficacy of TMCB132 in T cell-humanized mice. DETAILED DESCRIPTION OF THE INVENTION

[0024] All patents, patent applications, and related publications cited herein, including but not limited to: publications are incorporated herein by reference as if fully set forth.

[0025] The terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting. It should be understood that this is not intended to be a general term. All technical and scientific terms used herein are understood to be within the meaning of the present invention and are understood to be within the meaning of the present invention. has the same meaning as understood.

[0026] Any methods and materials similar or equivalent to those described herein may be used in testing the present invention. Although exemplary materials and methods can be used to perform the method, they are described herein. In describing and claiming the present invention, the following terminology will be used.

[0027] As used in this specification and the appended claims, "a," "an," and the singular "the" refers to a plural unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes two or more This includes combinations of the above cells, and the like.

[0028] "Specific binding" or "specifically binds" or "binds" means that an antibody binds to an antigen or This refers to the ability of a specific antigen to bind to an epitope within the antigen with higher affinity than other antigens. Typically, the antibody is approximately 5 x 10 -8 M or less, e.g., about 1×10 -9 M or less, approximately 1×10 -1 0 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ) binds to the antigen or epitope within the antigen, and non-specific antigens (e.g., BSA, casein) For binding to D At least 100 times smaller than K D Join with Dissociation constants can be measured using standard techniques. Antibodies that specifically bind to a target antigen may also bind to other related antigens, e.g., human or monkey, e.g., M acaca fascicularis (cynomolgus monkey, cyno) or Pan tro The same antigen (homologue) from other species, such as the glodytes (chimpanzee) Monospecific antibodies may have cross-reactivity to one antigen or one enzyme. Bispecific antibodies bind specifically to two different antigens or two different targets. They specifically bind to different epitopes.

[0029] "Antibody" has a broad meaning and includes murine, human, humanized and chimeric monoclonal antibodies. monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimers, Tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and other antibodies are also available. and any other modified configuration of immunoglobulin molecules containing an antigen-binding site of the desired specificity. "Full-length antibody molecules" include those that are interlinked by disulfide bonds. Two heavy chains (HC) and two light chains (LC) are connected to ), and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (heavy chain variable region, VH), and heavy chain constant region (domain CH1, Each light chain is composed of a light chain variable region (CH1, CH2, and CH3). It consists of a light chain constant region (CL) and a light chain variable region (VL). The VH and VL regions are composed of complementarity-determining regions interspersed with framework regions (FR). Each VH and VL can be further subdivided into regions of hypervariability called CDRs. From amino-terminus to carboxy-terminus, the order is: FR1, CDR1, FR2, CDR 2, FR3, CDR3, and FR4, It consists of

[0030] A "complementarity determining region (CDR)" is the region of an antibody that binds to an antigen. bat(Wu et al. (1970) J Exp Med 132:211-50) (Kabat et al., Sequences of Proteins of I mmunological Interest,5th Ed.Public Heal th Service,National Institutes of Health , Bethesda, Md., 1991), Chothia (Chothia et a (1987) J Mol Biol 196:901-17), IMGT (Lefr anc et al. (2003) Dev Comp Immunol 27:55-7 7), and AbM (Martin and Thornton (1996) J Bmol Biol 263:800-15) The correspondence between the various descriptions and the numbering of the variable regions is given (e.g., Lefranc et al.(2003)Dev Comp Immunol 27:55-77;H onegger and Pluckthun, (2001) J Mol Biol 3 09:657-70;International ImMunoGeneTics(I MGT) database; web source, see http: / / www_imgt_org (This refers to available programs such as abYsis by UCL Business PLC.) A CDR can be delineated using a gram. As used herein, the term "CDR" DR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCD Unless otherwise expressly stated in the specification, "LCDR2", "LCDR3", and "LCDR4" are the same as those in the above Kab CDRs defined by any of the methods of AT, Chothia, IMGT, or AbM Includes:

[0031] Immunoglobulins are divided into five major classes, depending on the amino acid sequence of the heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM can be assigned to IgA and IgD. IgG isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and Ig The antibody light chains of any vertebrate species are further subdivided as G4 and G5. Based on amino acid sequence, there are two distinct types: kappa (κ) and lambda (LAMBDA). can be assigned to one of the two λ.

[0032] "Antigen-binding fragment" means a portion of an immunoglobulin molecule that binds to an antigen. The fragment may be a synthetic polypeptide, an enzymatically obtainable polypeptide, or a genetically engineered polypeptide. and may be a polypeptide comprising VH, VL, VH and VL, Fab, F(ab'), F d and Fv fragments, domain antibodies consisting of one VH domain or one VL domain (d Ab), shark variable IgNAR domain, camelized VH domain, FR3-CDR3-F The smallest recognition unit, HCD, consists of amino acid residues that replicate the CDR of an antibody, such as the R4 portion. R1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3. The VH and VL domains are linked to each other via a synthetic linker. Various types of single chain antibody designs can be formed, in which the VH and VL domains are separated When expressed by a single chain antibody construct, the VH / VL domains are separated intramolecularly. Alternatively, the molecules may be paired to form a monovalent antigen-binding site, such as a single chain Fv (single chain Fv). cFv) or diabodies, which are described, for example, in WO 1998 / 44001, 1988 / 01649, 1994 / 13804, and 1992 / 01047.

[0033] A "monoclonal antibody" refers to a substantially homogeneous population (i.e., a population) of antibody molecules. Individual antibodies may be modified by removing the C-terminal lysine from the antibody heavy chain or by amino acid isomerization or alpha modification. Amidolysis, methionine oxidation, or asparagine or glutamine amidation "antibody" refers to an antibody obtained from a human (which is identical except for possible known modifications such as post-translational modifications) Monoclonal antibodies typically bind to one antigenic epitope. Monoclonal antibodies bind to two different antigenic epitopes. may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies are monospecific or may be multispecific, such as bispecific, monovalent, bivalent, or multivalent It may be.

[0034] An "isolated antibody" is an antibody or antibodies that are substantially free of other antibodies having different antigen specificities. (For example, an isolated antibody that specifically binds an antigen may be fragmented into fragments specific for antigens other than that antigen.) In the case of a bispecific CD3 antibody, the bispecific antibody An "isolated antibody" is an antibody that is 80% pure and specifically binds to both CD3 and a second antigen. ,81%,82%,83%,84%,85%,86%,87%,88%,89%,90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 This includes antibodies isolated to a high degree of purity, such as 00% pure antibodies.

[0035] A "humanized antibody" is an antibody in which at least one CDR is derived from a non-human species and at least one A humanized antibody refers to an antibody in which the framework is derived from a human immunoglobulin sequence. The framework can contain substitutions so that the framework is compatible with expressed human immunoglobulins. may not be an exact copy of the immunoglobulin or human immunoglobulin germline gene sequence. do.

[0036] A "human antibody" is an antibody that is optimized to provoke a minimal immune response when administered to a human subject. Human antibodies refer to antibodies that are humanized. The variable regions of human antibodies are derived from human immunoglobulin sequences. When a human antibody contains a constant region or a portion of a constant region, the constant region is also a human immunoglobulin Human antibodies are antibodies in which the variable regions of the human antibody are derived from human germline immunoglobulin or recombinant human antibody sequences. When derived from a system that uses assembled immunoglobulin genes, the sequence is "derived" from human origin. An exemplary such system comprises a heavy chain variable region and a light chain variable region that are derived from a phage. Displayed human immunoglobulin gene library and human immunoglobulin gene The present invention also includes transgenic non-human animals, such as mice or rats, carrying the locus. "Antibody" typically refers to the system used to obtain human antibodies and human immunoglobulin loci. differences in the amino acid sequence, the introduction of somatic mutations or substitutions into the framework or CDRs or both, resulting in amino acid substitutions when compared to human-expressed immunoglobulins. Typically, a "human antibody" is a human antibody derived from human germline immunoglobulins or rearranged antibodies. The amino acid sequence encoded by the immunoglobulin gene is The sequence of the amino acid is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% identical. In some cases, a "human antibody" is a human antibody, e.g., Pik et al. (2000) J Mol Biol 296:57-86 Consensus framework sequences derived from analysis of known human framework sequences, or For example, Shi et al. (2010) J Mol Biol 397:385- 96 and WO 2009 / 085462, containing a synthetic HCDR3 integrated into a human immunoglobulin gene library It's fine.

[0037] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence refers to the percentage of sequence identity. It is important to take into account all conservative substitutions as part of the sequence identity so that the percent identity is maximized. After aligning the sequences and introducing gaps if necessary, It is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the base sequence. Alignment for purposes of determining percent amino acid sequence identity is performed according to the principles of the art. Various methods within the skill of the art, e.g., BLAST, BLAST-2, ALIG N, or publicly available software such as Megalign (DNASTAR) software. This can be achieved using computer software. including any algorithms necessary to achieve maximal alignment over time, Appropriate parameters for aligning sequences can be determined. For purposes of this specification, the sequence comparison computer program ALIGN-2 was used. The ALIGN-2 sequence comparison computer program generates percent amino acid sequence identity values. The ram was created by Genentech, Inc. and the source code is protected by the U.S. Copyright Office. (Washington DC, 20559) along with user documentation. Registered under U.S. Copyright Registration Number TXU510087. ALIGN-2 Program Genentech.Inc. (South San Francisco, Ca. lif.) or may be compiled from source code. The ALIGN-2 program is designed to support UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters must be compiled for use in the operating system. is set by the ALIGN-2 program and remains unchanged. When ALIGN-2 is used for a given amino acid sequence B, a given alignment is performed for The % amino acid sequence identity of amino acid sequence A to a given amino acid sequence B (or to a given amino acid sequence B) A given amino acid sequence A has or contains a particular amino acid sequence A that is identical to (which can be calculated as follows: 100 x ratio X / Y, (wherein X is the sequence of A and B aligned by the sequence alignment program ALIGN-2) The number of amino acid residues scored as perfect matches by the program in the statement and Y is the total number of amino acid residues in B). If the length of string B is not equal, then the % amino acid sequence identity of A to B is It will be understood that this does not equate to percent amino acid sequence identity. All % amino acid sequence identity values ​​used herein are calculated using the ALIGN-2 computer The program is obtained as described in the immediately preceding paragraph.

[0038] Antibodies whose antigen-binding site is derived from a non-human species are not included in the definition of "human antibody."

[0039] "Recombinant" refers to a recombinant DNA, antibody, or is prepared, expressed, produced, or isolated by recombinant means when producing a protein Refers to DNA, antibodies, and other proteins.

[0040] "Epitope" refers to the part of an antigen to which an antibody specifically binds. Typically, chemically active (polar, non-polar or hydrophobic) moieties such as amino acids or polysaccharide side chains Epi- and other surface groups may have specific charge characteristics in addition to specific three-dimensional structural characteristics. A topope is composed of consecutive and / or discontinuous amino acids that form a conformational spatial unit. In a discontinuous epitope, amino acids that are in different parts of the linear sequence of the antigen Protein molecules fold together to bring them into close proximity in three-dimensional space. The "type" varies depending on the methodology used to identify the epitope.

[0041] "Paratope" refers to the portion of an antibody that specifically binds to an antigen. It may be a linear series of amino acids, or it may be a non-contiguous amino acid sequence of an antibody, rather than a linear series of amino acids. It may be discontinuous, formed by the spatial relationship of the acids. "paratope" and "heavy chain paratope" or "amino acid residues of light chain paratope" and "heavy chain The "amino acid residues of the paratope" respectively represent the light chain residues and heavy chain residues of the antibody that come into contact with the antigen. "Antibody paratope residues" refers to, or generally refers to, those antibody amino acids that contact the antigen. Refers to...

[0042] "Bispecific" refers to a molecule that is specific to two different antigens or two different epitopes on the same antigen. Bispecific antibodies refer to antibodies that bind to different antigens. Bispecific antibodies can bind to different antigens, e.g., human or monkey, For example, the same antigen from another species, such as cynomolgus monkey (cyno) or chimpanzee (homologous) may have cross-reactivity to other antigens (log) or may be shared between two or more different antigens The antibody can bind to an epitope that is present in the antibody.

[0043] "Multispecific" refers to the ability to bind two or more different antigens within the same antigen, or two or more different epitopes. A multispecific antibody refers to an antibody that specifically binds to a specific antigen, e.g., a fusion protein. from humans or other species such as monkeys, e.g., cynomolgus monkeys (cyno) or chimpanzees They may have cross-reactivity to the same antigen (homologs) or to two or more different antigens It can bind to an epitope shared between the two.

[0044] A "variant" is a member of a family of amino acids that has been modified by one or more alterations, e.g., one or more substitutions, insertions, or deletions. A polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide. Refers to...

[0045] A "vector" is an organism capable of replicating within a biological system or being transferred between such systems. A vector polynucleotide typically refers to a polynucleotide that replicates a vector. Using biological elements that can be used to create living systems such as cells, viruses, animals, and plants, These polynucleotides have the function of promoting the replication or maintenance of these polynucleotides in the reconstituted biological system. It contains elements such as an origin of replication, a promoter, a polyadenylation signal, and a selection marker. A vector polynucleotide may be a single-stranded or double-stranded DNA or RNA molecule. , or a hybrid thereof.

[0046] An "expression vector" is a vector encoding a polypeptide that is encoded by a polynucleotide sequence present in the expression vector. used in a biological system or a reconstituted biological system to direct the translation of a polypeptide to be Refers to vectors that can be used.

[0047] "Polynucleotide" means a molecule that is shared by a sugar-phosphate backbone or other equivalent covalent bonding chemistry. refers to molecules containing chains of linked nucleotides. Double-stranded and single-stranded DNA and RNA is a typical example of a polynucleotide. Contains a chain of nucleotides linked by covalent or other equivalent covalent chemical reactions It may refer to a synthetic molecule, and cDNA is an exemplary synthetic polynucleotide.

[0048] A "polypeptide" or "protein" is a molecule that is linked by peptide bonds to form a polypeptide. It refers to a molecule containing at least two amino acid residues that form a bond. Small polypeptides consisting of these may be referred to as "peptides."

[0049] "Flow cytometry" refers to the measurement of a fluid as it passes through at least one laser. It is a technique used to analyze the physical and chemical properties of particles in the body. Fluorescently labeled and then excited by a laser to emit light at various wavelengths (Adan, et al,Critical Reviews in Biotechnology( 2016)1549-7801).

[0050] An "anti-idiotypic (anti-Id) antibody" is an antibody that binds to an antigenic determinant (e.g., a paratope or The process of producing or preparing an anti-idiotype antibody is as follows: It is generally known in the art. (Lathey, J. et al. Immun ology 1986 57(1):29-35). Anti-Id antibodies block the antigen. Antigen-blocking anti-Id antibodies may or may not be This can be used to detect free antibodies, such as CD3, in a sample. The non-binding anti-Id antibody is the total antibody in the sample (free, partially bound to antigen). can be used to detect antibodies that are either fully or completely bound to the antigen. Anti-Id antibodies are prepared by immunizing animals with antibodies against which anti-Id antibodies have been prepared. In some embodiments described herein, anti-idiotype The antibody is used to detect the level of the therapeutic antibody in the sample.

[0051] In addition, anti-Id antibodies have been used as immunogens to induce immune responses in other animals, resulting in the so-called It is also possible to produce anti-anti-Id antibodies that are induced by the original anti-Id antibody. The mAb and the epitope may be identical. By using antibodies that express the same specificity, other clones can be identified. The anti-Id antibody may be varied (thereby allowing for anti-Id antibody variants). produced), and / or as described elsewhere herein with respect to anti-CD3 antibodies. The derivatives may be derivatized by any suitable technique, such as by

[0052] PSMA refers to prostate-specific membrane antigen. The amino acid sequence of PSMA (also called chimp or chimp) is shown in SEQ ID NO: 49. The extracellular domain spans residues 44-750 of SEQ ID NO: 49, and the transmembrane domain spans residues 2 The cytoplasmic domain spans residues 0–43, and the cytoplasmic domain spans residues 1–19. scicularis (also called cynomolgus monkeys, macaques, or cynos) PSMA The amino acid sequence is shown in SEQ ID NO: 50. The extracellular domain is composed of residues 44 to 75 of SEQ ID NO: 50. 0, the transmembrane domain spans residues 20–43, and the cytoplasmic domain spans residues The amino acid sequence of human PSMA is shown in SEQ ID NO: 51. The transmembrane domain spans residues 44-750 of SEQ ID NO: 51, and the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1–19.

[0053] Throughout this specification, the terms "CD3 specific" or "specifically binds to CD3" or "antibody" are used. "CD3 antibody" specifically binds to the CD3-ε extracellular domain (ECD) (SEQ ID NO: 636). Antibodies that specifically bind to CD3-ε polypeptide (SEQ ID NO: 635), including antibodies that bind to CD3-ε refers to CD3-γ, -δ, and -ζ, as well as T cell receptors α / β and γ. Together with the CD3 / δ heterodimer, they form the T cell receptor-CD3 complex. It plays an important role in linking antigen recognition to several intracellular signaling pathways. The D3 complex mediates signal transduction, resulting in T cell activation and proliferation. 3 is required for immune responses.

[0054] SEQ ID NO: 635 (human CD3ε) MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVS ISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVC ENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKA KPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI

[0055] SEQ ID NO: 636 (human CD3ε extracellular domain) DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPR GSKPEDANFYLYLRARVCENCMEMD

[0056] As used herein, the term "interleukin-1 receptor accessory protein" refers to a protein that is a protein that binds to an interleukin-1 receptor. "," "IL1RAP," and "IL1-RAP" are specifically listed in, for example, GenBan k Accession No. AAB84059, NCBI Reference S sequence:NP_002173.1, and UniProtKB / Swiss-Pr and human IL-1 as described in Accession No. Q9NPH3-1. 1RAP protein (Huang et al., 1997, Proc. Natl. See also Acad.Sci.USA.94(24),12829-12832. IL1 RAP has also been linked to IL1 R3, C3orf13, FLJ37788, and I in the scientific literature. Also known as L-1 RAcP and EG3556.

[0057] "CD33" refers to the cluster of differentiation 33 protein. CD33 is a 67 kilodalton (kD) single-pass transmembrane glycoprotein, and sialic acid-binding immunoglobulin-like lectin (sialic acid CD33 is a member of the Glc family. It is primarily considered to be a myeloid differentiation antigen. It is expressed at low levels in myeloid progenitor cells, neutrophils, and macrophages, and in circulating monocytes and dendritic cells. Human CD33 extracellular domain (Uniprot P20138) (SEQ ID NO: No. 636) and cynomolgus monkey CD33 (XP_005590138.1) are used in accordance with the present disclosure. are examples of proteins for use in generating CD33-specific antibodies.

[0058] "TMEFF2" contains two folio domains, an EGF-like domain and a domain called tomoregulin 2. It refers to a human transmembrane protein containing statin-like domain 2. The amino acid sequence is shown in SEQ ID NO: 77. The extracellular domain of TMEFF2 is shown in SEQ ID NO: 575. The TMEFF2 extracellular domain spans residues 40–374 of the full-length TMEFF2. , three distinct subdomains, Kazal-like 1 (residues 85–137), Kazal-like 2 ( It contains the TME domain (residues 176-229) and the EGF domain (residues 261-301). The FF2 EGF domain is shown in SEQ ID NO: 577. The TMEFF2 "membrane proximal region" is The F domain and the NC-terminal linker region (e.g., full-length human TMEFF of SEQ ID NO: 77) This refers to the TMEFF2 region of SEQ ID NO: 629, encompassing residues 230-320 of SEQ ID NO: 629. All references in the specification to proteins, polypeptides, and protein fragments are to non- Unless explicitly designated as being of human species origin, each protein, polypeptide, The term "TM" is intended to refer to a human version of a protein, peptide, or protein fragment. "TMEFF2" refers to a gene encoding a TMEFF2 protein from a non-human species, such as "mouse TMEFF2" or "monkey TMEFF2." Unless specified as being from any non-human species, human TMEFF2 is meant.

[0059] SEQ ID NO: 77 (full length human TMEFF2) MVLWESPRQCSSWTLCEGFCWLLLLPVMLLIVARPKLA AFPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGE CLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQA ACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKET STCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDG KSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSED GHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHKCE HSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQY VLIAAVIGTIQIAVICVVVLCITRKPRSNRIHRQKQNTG HYSSDNTTRASTRLI

[0060] SEQ ID NO: 575 (extracellular domain of human TMEFF2) FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGE CLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQA ACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKET STCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDG KSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSED GHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHKCE HSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQY VLIAAVIGTIQIAVICVVVLCITRKPRSNRIHRQKQNTG HYSSDNTTRASTRLI

[0061] TMEFF2 EGF domain SEQ ID NO: 577 HHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQH CE

[0062] TMEFF2 membrane proximal region SEQ ID NO: 629 NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEH YNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVL YVVPGPVRFQYV

[0063] "TMEFF2-positive cancer" is cancer tissue that exhibits measurable levels of TMEFF2 protein. or cancer cells. TMEFF2 protein levels are measured in the periphery of live or lysed cells. known assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassays. The term "overexpression," "overexpressed," and "overexpressed" can be used to describe the expression of a protein. "Overexpressed" interchangeably means a measurably higher level when compared to a reference sample. The term "overexpression" refers to a sample of cancer cells, malignant cells, or cancer tissues that have a tumor antigen of It can be caused by gene amplification or by increased transcription or translation. Expression and overexpression of proteins in cells can be achieved using well-known assays for live or lysed cells, for example. Assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay The expression and overexpression of polynucleotides in a sample can be measured using the method described above. For example, fluorescent in situ hybridization, Southern blotting, or P The amount of protein or polynucleotide in the sample can be measured using CR technology. A protein or peptide is considered to be a nucleotide analog when its level is at least 1.5-fold higher than that of the reference sample. The polynucleotide is over-represented. The selection of a reference sample is well known.

[0064] A "sample" refers to any fluid, cell, or tissue isolated from a subject, as well as any other fluid, cell, or tissue isolated from a subject. A sample refers to a collection of fluids, cells, or tissues present within a body. Exemplary samples include blood, serum, and Body fluids and secretory tissues such as serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, and sputum and secretions from mucous membranes of organs, vaginal secretions, ascites such as those associated with non-solid tumors, and pleural cavities. Fluids from the pericardial cavity, peritoneal cavity, abdominal cavity, and other body cavities; fluids recovered by bronchial lavage; Culture media for animals or biological sources, such as cells and organs (including conditioned media for cells or organs) liquid solutions that have come into contact with tissue biopsies, fine needle aspirations, or surgically removed tumor tissue. It is a weave.

[0065] As used herein, a "cancer cell" or "tumor cell" refers to a cell that is grown in vivo, ex vivo, or In tissue culture, cancerous cells with spontaneous or induced phenotypic changes are These changes do not necessarily involve the incorporation of new genetic material. Transformation does not involve infection with a transforming virus and the assembly of new genomic nucleic acid. It can occur spontaneously or in response to carcinogens, but can also occur through the incorporation of exogenous nucleic acids or the uptake of exogenous nucleic acids. It can also occur after exposure to a pathogenic agent, which results in mutations in endogenous genes. The gene is capable of inducing morphological changes, cell immortalization, and differentiation in vitro, in vivo, and ex vivo. Regulation of normal proliferation, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, and invasion and tumor growth in suitable animal hosts such as nude mice (Fr eshney,Culture of Animal Cells:A Manual of Basic Technique (3rd ed., 1994).

[0066] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges, described herein are In all cases, the terms "about" and "about" should be understood as being modified. Therefore, numerical values ​​typically 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, 1% to 10 The concentration range of % (w / v) includes 0.9% (w / v) to 11% (w / v). When used, the use of a numerical range shall be interpreted as meaning that the range is true unless the context clearly indicates otherwise. All possible subranges, including integers and fractions of values, and all individual numbers within that range Explicitly included.

[0067] "Effector antigens" are antigens that mediate cytotoxicity, phagocytosis, antigen presentation, and / or cytokine release. These effects are antigens that can stimulate or induce the release of antibodies from cells of the immune system. Target antigens include, but are not limited to, T cells and natural killer (NK) cells. Examples of suitable specificities for effector antigens include CD3 for T cells and CD4 for CD8. CD3 subunits such as CD3ε and CD16 for NK cells, Such cell surface molecules of effector cells mediate cell killing. Effector cells are suitable for cytotoxicity, phagocytosis, antigen presentation, and / or These are cells of the immune system that can stimulate or induce cytokine release. Vector cells include, for example, T cells, natural killer (NK) cells, granulocytes, monocytes, macrophages, and Effectors include, but are not limited to, phage, dendritic cells, and antigen-presenting cells. Examples of suitable specificities of T cells include, but are not limited to: For cells, CD2, CD3, and CD3 subunits such as CD3e, CD5, CD28 , and other components of the T cell receptor (TCR); for NK cells, CD16, CD16 A, CD25, CD38, CD44, CD56, CD69, CD94, CD335(NK p46), CD336, (NKp44), CD337(NKp30), NKp80, NK G2C and NKG2D, DNAM, NCR; and CD18, CD64, and CD89; CD18, CD32, CD64, and CD89 for monocytes and macrophages and mannose receptor for dendritic cells; CD64 and mannose receptor for dendritic cells; and C D35. In certain embodiments of the present invention, the specificity of effector cells, i.e., cell surface moieties, The molecule induces cell killing upon binding of the bispecific or multispecific molecule to such cell surface molecules. It is suitable for mediating the cytotoxicity of ATP and thereby inducing cell lysis or apoptosis.

[0068] A "bispecific CD3 antibody" is a compound that contains at least one binding domain that specifically binds to CD3. and a molecule comprising at least one binding domain that specifically binds to a second antigen, e.g. a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen, This refers to a bispecific antibody that contains a domain that specifically binds to CD3 and a second antigen. is typically V H / V L A bispecific CD3 antibody binds to either CD3 or a second antigen. In some embodiments, the second or target may be monovalent with respect to binding to either Target antigens are cell surface antigens expressed on target cells other than immune effector cells. In some embodiments, the second antigen is a tumor-associated antigen (TAA). Exemplary TAAs include: , PSMA, CD33, IL1RAP, and TMEFF2.

[0069] “Bispecific PSMA×CD3 antibody”, “PSMA / CD3 antibody”, “Bispecific anti-P "SMA × CD3 antibody" or "anti-PSMA / CD3 antibody" specifically binds to PSMA. at least one binding domain that specifically binds to CD3; and at least one binding domain that specifically binds to CD3. The domains that specifically bind to PSMA and CD3 are typically In terms of type, V H / V L The bispecific anti-PSMA×CD3 antibody is a pair. 3. It may be monovalent with respect to attachment to any of the three.

[0070] “Bispecific CD33×CD3 antibody”, “CD33 / CD3 antibody”, “bispecific anti-C "CD33 × CD3 antibody" or "anti-CD33 / CD3 antibody" specifically binds to CD33. at least one binding domain that specifically binds to CD3; and at least one binding domain that specifically binds to CD3. The domain that specifically binds to CD33 and CD3 is typically In terms of type, V H / V L The bispecific anti-CD33×CD3 antibody binds to either CD33 or CD3. 3. It may be monovalent with respect to attachment to any of the three.

[0071] “Bispecific IL1RAP×CD3 antibody”, “IL1RAP / CD3 antibody”, “bispecific IL1RAP×CD3 antibody” "Isomeric anti-IL1RAP × CD3 antibody" or "anti-IL1RAP / CD3 antibody" are At least one binding domain that specifically binds to 1RAP and at least one binding domain that specifically binds to CD3 It refers to a molecule containing at least one binding domain specific to IL1RAP and CD3. The domain that binds to V H / V L Paired. Bispecific anti-IL1RAP×C The D3 antibody may be monovalent with respect to binding to either IL1RAP or CD3.

[0072] "Bispecific anti-TMEFF2 / anti-CD3 antibody", TMEFF2 / CD3 antibody, TMEF F2xCD3 antibody and the like refer to an antibody that binds to TMEFF2 and CD3.

[0073] "Valency" refers to the specified number of specific binding sites for an antigen in a molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" respectively mean: This refers to the presence of one, two, four, or six specific binding sites for an antigen in a molecule. "Multivalent" refers to the presence of two or more specific binding sites for an antigen in a molecule. vinegar.

[0074] "Antigen-specific CD4+ or CD8+ T cells" are CD4+ or CD8+ T cells activated by a specific antigen. It refers to CD4+ or CD8+ T cells, or their immunostimulatory epitopes.

[0075] A "subject" includes any human or non-human animal. A "non-human animal" includes, for example, mammals. Mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens This includes all vertebrates, including mammals, amphibians, and reptiles. The terms "person" and "subject" are used interchangeably.

[0076] Throughout this specification, the numbering of amino acid residues in the constant region of an antibody is specifically Unless otherwise specified, Kabat et al., Sequences of Proteins ns of Immunological Interest,5th Ed.Publ ic Health Service,National Institutes of Health, Bethesda, MD. (1991) Comply with the standard.

[0077] [Table 1]

[0078] Material composition The present invention relates to anti-CD3 antibodies and antigen-binding fragments thereof, antibodies that specifically bind to PSMA, and Antibody specifically binding to CD33 and antigen-binding fragment thereof, IL1RA an antibody or antigen-binding fragment thereof that specifically binds to P; a first domain that specifically binds to CD3; a multispecific antibody comprising a second domain that specifically binds to a second antigen and a C Specific for D3 and one or more of PSMA, CD33, IL1RAP, and TMEFF2 The present invention provides a multispecific antibody that specifically binds to a target antigen. Encoding polypeptides and polynucleotides, vectors, host cells, and Methods of making and using the compound are provided.

[0079] General Aspects of the Antibodies Described Herein The anti-CD3 specific antibodies or antigen-binding fragments described herein are the same as those described anti-CD3 antibodies or retains the biological properties (e.g., binding affinity or immune effector activity) of the antigen-binding fragment The present invention also includes variants having one or more amino acid substitutions, deletions, or additions. In the present context, the following notation is used to describe the mutations unless otherwise stated. i) An amino acid substitution at a given position is described as, for example, K409R. R means substitution of arginine for lysine at position 409. ii) Specific Mutants For the specific three-letter or one-letter codes, including the codes Xaa and X, the amino acid residues Therefore, the substitution of arginine for lysine at position 409 is The substitution is designated K409R, or any amino acid residue for the lysine at position 409. The substitution of this residue is designated K409X. In the case of a deletion of the lysine at position 409, The deletion is K409 * Those skilled in the art will recognize that one or more amino acid substitutions, deletions, Mutants with deletions or additions can be produced.

[0080] These variants include (a) one or more amino acid residues that are conservative or non-conservative; (b) variants in which one or more amino acids are substituted by a specific amino acid; (c) one or more variants in which one or more amino acids are added to or deleted from the polypeptide; (d) variants in which the amino acids of the polypeptide are substituted with another peptide or polypeptide; tides, e.g., variants fused to fusion partners, protein tags, or other chemical moieties; These can be used to give the polypeptide useful properties, such as antibody binding. The nucleic acid may be provided with a nucleotide sequence, a nucleotide sequence, a polyhistidine sequence, a biotin moiety, or the like. The antibody or antigen-binding fragment may contain certain amino acids at either conserved or non-conserved positions. may include variants in which amino acid residues from the In other embodiments, the amino acid residues at the non-conserved positions are either conserved or non-conserved residues. These mutants are obtained by genetic (deletions, mutations, etc.), chemical These methods, including chemical and enzymatic methods, are known to those skilled in the art.

[0081] The antibodies or antigen-binding fragments described herein may be IgM, IgD, IgG, IgA, or IgM. In some embodiments, the antibody isotype is IgG. In some embodiments, the antibody is of the IgG1, IgG2, IgG3, or IgG4 isotype. 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 specificity of is determined in part by the amino acid sequence and arrangement of the CDRs. The CDRs of one isotype can be transferred to another isotype without changing the antigen specificity. Such antibody isotypes can therefore be used in combination with the described antibodies or Within the scope of antigen-binding fragments.

[0082] The IgG class has four isotypes in humans: IgG1, IgG2, and IgG3. These isotypes are divided into IgG1, IgG2, and IgG4. The amino acid sequence of the hinge region and the ribosomal region share over 95% homology. The Fc region is involved in effector functions, e.g., antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cellular injury In ADCC, the Fc region mediates immune effector cells, e.g., It binds to Fc receptors (FcγR) on the surface of natural killer cells and macrophages. In CDC, antibodies block the complement cascade on the cell surface, causing lysis of the target cell. In ADCP, antibodies mediate the killing of target cells by inducing the Mediates elimination of antibody-coated target cells by internalization by phagocytic cells such as leukocytes or dendritic cells The antibodies described herein include antibodies in which the Fc region regulates various effector functions. in combination with any of the IgG isotypes, including modified versions Antibodies having the described characteristics of the variable domains are included.

[0083] Many applications of therapeutic antibodies potentially pose safety risks due to cell depletion. Therefore, Fc-mediated effector functions are undesirable. This can be achieved by genetically engineering regions to reduce binding to FcγRs or complement factors. . Activation (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and Binding of IgG to inhibitory (FcγRIIb) FcγR or the first component of complement (C1q) Depends on residues located in the hinge region and CH2 domain. IgG1, IgG2, and I Mutations can be introduced into gG4 to reduce or silence Fc-mediated effector functions. The antibodies described herein may include these modifications.

[0084] In some embodiments, anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1R The AP antibody comprises an engineered Fc region with one or more of the following properties: a) reduced effector function compared to parent Fc; (b) FcγRI, FcγRI Ia, FcγRIIb, FcγRIIIb, and / or FcγRIIIa (c) reduced affinity for FcγRI; (d) reduced affinity for FcγR (e) reduced affinity for FcγRIIIb; or (f) has reduced affinity for FcγRIIIa.

[0085] In some embodiments, anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1R The AP antibody may be, for example, an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody has an IgG4 isotype, and the antibody has a wild-type IgG 4 isotype, S228P, F234A, and L235A in its Fc region. In some embodiments, the antibody has an IgG1 isotype and contains a substitution. , which contains L234A and L235A substitutions in its Fc region. The body may contain these modifications.

[0086] In some embodiments, anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1R The AP antibody is of the IgG4 isotype, optionally containing the heavy chain substitution S228P.

[0087] In some embodiments, anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1R The AP antibody optionally contains heavy chain substitutions L234A, G237A, It is an IgG1 isotype containing P238S, H268A, A330S, and P331S. do.

[0088] In some embodiments, anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1R The AP antibody optionally contains heavy chain substitutions L234A, G237A, IgG2 antibody containing P238S, H268A, V309L, A330S, and P331S It is a sotype.

[0089] Wild type IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 602)

[0090] Wild type IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 601)

[0091] Wild type IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 711)

[0092] In certain embodiments, labeled anti-CD3, anti-PSMA, anti-CD33, and / or anti-I L1RAP antibodies are provided. Exemplary directly detectable labels or moieties (e.g., fluorescent, luminescent) chromophores, electron-dense, chemiluminescent, and radioactive labels) and (e.g., enzymatic reactions or intermolecular interactions Labels and moieties (e.g., enzymes or ligands) that are indirectly detected (through interaction). Suitable labels include radioactive labels (e.g.,32 P, 14 C. 111 I, 125 I, 3 H, 131 I), fluorescent labels (such as DyLight® 649), epitope tags, The label may be a chromophore label, an ECL label, or an enzyme. Ruthenium, 111 In-DOTA, 111 In-diethylenetriamine penta Acetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and beta Galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, Luoron dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Alexaf Luor® dyes and the like.

[0093] The anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1RAP antibodies described herein, and In addition to antigen-binding fragments thereof, polynucleotides capable of encoding the described antibodies and antigen-binding fragments are also provided. Also provided are vectors containing the described polynucleotides, as well as , anti-CD3, anti-PSMA, anti-CD33, and / or anti-IL1R antibodies provided herein Cells expressing the AP antibodies or antigen-binding fragments are also provided. Also described are cells capable of expressing the nucleotide sequence nucleotides 1 and 2. These cells include mammalian cells (e.g., 293F cells). , CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells The described antibodies may also be produced by hybridoma cells (e.g., E. coli). It can be produced.

[0094] Generation of monospecific antibodies In some embodiments, the anti-CD3 antibodies of the invention, anti-PSMA, anti-CD33, anti-TME The FF2 and / or anti-IL1RAP antibody is human.

[0095] In some embodiments, the anti-CD3 antibodies of the invention, anti-PSMA, anti-CD33, anti-TME FF2 and / or the anti-IL1RAP antibody is humanized.

[0096] The monospecific antibodies of the invention described herein (e.g., anti-CD3, anti-PSMA, anti-CD3 3, anti-TMEFF2, and / or anti-IL1RAP antibodies) can be generated using a variety of techniques. See, for example, Kohler and Milstein, Nature 256:4 Monoclonal antibodies can be produced using hybridoma techniques such as those used in the 1975 and 1980s. In the hybridoma method, mice or other host animals, such as hamsters, rats, or Chickens were transfected with human, chimpanzee, or macaque PSMA, CD33, IL1RAP, and T MEFF2, or CD3, or PSMA, CD33, IL1RAP, TMEFF2, or fragments of CD3, e.g., PSMA, CD33, IL1RAP, TMEFF2, or the extracellular domain of CD3, followed by immunoprecipitation using standard methods. Spleen cells from the animals were fused with myeloma cells to form hybridoma cells (God ing, Monoclonal Antibodies: Principles and Practice,pp.59-103(Academic Press,1986) Colonies generated from a single immortalized hybridoma cell were analyzed for binding specificity, cross-reactivity, and Screening is performed for the production of antibodies with desired properties, such as reactivity or lack thereof, and antigen affinity. To train.

[0097] A variety of host animals can be used to administer the anti-CD3, anti-PSMA, and anti-CD20 antibodies of the invention described herein. 33, TMEFF2, and / or anti-IL1RAP antibodies can be produced. For example, Balb / c mice can be used to generate mouse anti-human PSMA antibodies. Antibodies generated in alb / c mice and other non-human animals are used to generate more human-like antibodies. It can also be humanized using a variety of techniques to generate sequences.

[0098] Exemplary humanization techniques, including the selection of a human acceptor framework, are known and described in CD R grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6, 818,749), resurfacing (Padlan (1991) Mol Immun ol 28:489-499), resurfacing of specificity-determining residues (U.S. Patent Application Publication No. 2010 / 0261620), human framework adaptation (U.S. Patent No. 8,748,3 56), or hyperhumanization (U.S. Patent No. 7,709,226). Based on similarity in CDR length or identity of canonical structure, or a combination of these. In this case, human frameworks can be selected based on overall homology to the parent framework. The CDRs of the parent antibody are introduced into the target gene.

[0099] Humanized antibodies are described in WO 1090 / 007861 and WO 1992 / 22653. Fragments were altered to preserve binding affinity by techniques such as those described in by incorporating framework-supporting residues (backmutations) or by varying either the CDRs to improve the affinity of the antibody, for example, thereby enhancing its selection for a desired antigen. It may be further optimized to improve activity or affinity.

[0100] mice or rats that have human immunoglobulin (Ig) loci in their genome and generating human antibodies against target proteins using transgenic animals of the present invention. These are described, for example, in U.S. Pat. No. 6,150,584; International Publication Nos. 99 / 45962, 2002 / 066630, and 2002 / 4347 No. 8, No. 2002 / 043478, and No. 1990 / 04036, Lonber g et al(1994)Nature 368:856-9;Green et a l(1994)Nature Genet.7:13-21;Green & Jako bovits(1998)Exp.Med.188:483-95;Lonberg a nd Huszar (1995) Int Rev Immunol 13:65-93; Bruggemann et al.,(1991)Eur J Immunol 21 :1323- 1326;Fishwild et al.,(1996) Nat Bi otechnol 14:845-851;Mendez et al.,(1997) Nat Genet 15:146-156;Green(1999)J Immuno l Methods 231:11-23;Yang et al.,(1999)Ca ncer Res 59:1236-1243;Bruggemann and Tau ssig(1997) Curr Opin Biotechnol 8:455-458 The endogenous immunoglobulin loci of such animals may be disrupted or deleted, and homologous or by using non-homologous recombination, by using transchromosomes, or by using minigenes Genes are used to engineer at least one complete or partial human immunoglobulin locus. It can also be inserted into the genome of a living organism. neron_com), Harbor Antibodies(http: / / _ww w_harbourantibodies_com), Open Monoclonal Technology, Inc.(OMT)(http: / / _www_omtinc _net), KyMab(http: / / _www_kymab_com), Trian ni(http: / / _www.trianni_com) and Ablexis(ht Companies such as tp: / / _www_ablexis_com) use the above technologies to There are efforts to provide human antibodies targeted to selected antigens.

[0101] Human antibodies can be selected from phage display libraries, in which case the phage The phage may be a human immunoglobulin or a portion thereof, such as a Fab, a single chain antibody (scFv), or genetically engineered to express the variable regions of unpaired or paired antibodies (Knap pik et al.,(2000)J Mol Biol 296:57-86;Kr ebs et al.,(2001)J Immunol Meth 254:67-8 4;Vaughan et al.,(1996)Nature Biotechnol ogy 14:309-314;Sheets et al., (1998) PITAS (USA)95:6157-6162;Hoogenboom and Winter( 1991)J Mol Biol 227:381;Marks et al.,(19 91) J Mol Biol 222:581). The antibody of the present invention can be used, for example, in the treatment of Shi e t al., (2010) J Mol Biol 397:385-96 and International Publication No. As described in the publication of the 09 / 085462, the bacteriophage pIX coat protein Phage display expressing antibody heavy and light chain variable regions as fusion proteins The library can be isolated from human and / or cynomolgus PSMA, Screening for phage binding to CD33, IL1RAP, TMEFF2, or CD3 The resulting positive clones were further characterized and Fab was isolated from the lysates of the clones. , and can be expressed as full-length IgG. Such methods for isolating human antibodies The phage display method is described, for example, in U.S. Pat. Nos. 5,223,409 and 5,403 , No. 484, No. 5,571,698, No. 5,427,908, No. 5,580, No. 717, No. 5,969,108, No. 6,172,197, No. 5,885,7 No. 93, No. 6,521,404, No. 6,544,731, No. 6,555,31 Nos. 3, 6,582,915, and 6,593,081.

[0102] Preparation of immunogenic antigens and production of monoclonal antibodies can be performed using any method, including recombinant protein production. Immunogenic antigens can be purified proteins or Alternatively, the protein may be administered to animals in the form of whole cells or protein mixtures containing cell or tissue extracts. The antigen may be denominated in the animal's body from a nucleic acid encoding the antigen or a part thereof. A bo may be formed.

[0103] Generation and Use of Bispecific and Multispecific CD3 Antibodies The present invention relates to a method for detecting a CD3-specific antigen comprising administering to a subject a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. and a second domain that binds to the second antigen. It may be a tumor-associated antigen (TAA) or an antigen on a pathogenic cell.

[0104] The first domain specifically binds to CD3 and the second domain specifically binds to a second antigen. The present invention can be used to engineer bispecific and multispecific antibodies, including antibodies Exemplary anti-CD3 antibodies that can be used include those listed in Tables 7A and 7B, respectively. H / V L Distribution and CD3 antibodies comprising the sequences and heavy / light chain CDRs, as well as Tables 10 and 11 and accompanying text. For example, the genetically engineered variants described herein include those described in CD3 antibodies CD3B312, CD3B313, CD3B314, CD3B315, CD3 B316, CD3B317, CD3B337, CD3B373, CD3B376, CD3 CDRs and / or VH / VL domains of B389, CD3B450, and CD3B467 a first domain that specifically binds to CD3 and a second domain that specifically binds to an antigen using Bispecific and multispecific antibodies can be generated that comprise a second domain.

[0105] The CDRs and / or VH / VL domains of the anti-CD3 antibodies described herein may be used to identify antibodies specific to CD3. a first domain that specifically binds to a second antigen and a second domain that specifically binds to a second antigen. It can be incorporated into a bispecific antibody.

[0106] The CDRs and / or VH / VL domains of the anti-CD3 antibodies described herein are also used herein and and incorporating the PSMA-binding VH / VL domains listed in Table 23 into a bispecific antibody. The anti-CD3 antibodies and / or VH / VL domains described herein can be used in the methods described herein and and a bispecific antibody comprising the IL1RAP-binding VH / VL domains listed in Table 30. The CDRs and / or VH / VL domains of the anti-CD3 antibodies described herein can be and a bispecific antibody comprising the CD33-binding VH / VL domains described herein and in Table 38. For example, the PSMA antibody PSMB119, PSM B120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB 126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB 120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB 128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB 347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB The VH / VL domains of PSMB361, PSMB362, PSMB363, and PSMB365 can be used to generate bispecific PSMAxCD3 antibodies.

[0107] Exemplary TAAs are PSMA, CD33, TMEFF2, and IL1RAP. Exemplary multispecific PSMAxCD3, CD33xCD3, TMEF, and IFN-γ derivatives are provided herein. The F2×CD3 and IL1RAP×CD3 antibodies have a first domain that specifically binds to CD3. and a second antibody that specifically binds to PSMA, CD33, TMEFF2, or IL1RAP. The PSMAxCD3 domains are used to engineer bispecific PSMAxCD3 molecules. Exemplary anti-PSMA antibodies that can be used are those described herein, which heavy and light chain sequences, including but not limited to, the heavy and light chain sequences listed in Table 23 Use to engineer bispecific IL1RAP x CD3 molecules. Exemplary anti-IL1RAP antibodies that can be used include those described herein, which heavy and light chain variable region sequences, including but not limited to, the heavy and light chain variable region sequences provided in Table 35. The CD33xCD3 molecule may comprise a light chain variable region sequence. Exemplary anti-CD33 antibodies that can be used are those described herein. , including, but not limited to, the heavy and light chain variable region sequences provided in Table 43. The bispecific TMEFF2xCD3 molecule can comprise heavy and light chain variable region sequences. Exemplary anti-TMEFF2 antibodies that can be used to generate TMEFF2 include those described herein. These are the heavy and light chain variable region sequences provided in Table 59 and Tables 66-68. The sequences may include heavy and light chain variable region sequences, including but not limited to:

[0108] The bispecific antibodies generated may be fused to either CD3 or a second antigen. and / or for desired functional properties such as T cell-mediated killing of cells expressing a second antigen. can be tested.

[0109] The bispecific antibodies provided herein include antibodies having a full-length antibody structure.

[0110] A "Fab arm" or "half molecule" refers to one heavy chain-light chain pair that specifically binds to an antigen. means.

[0111] The full-length bispecific antibodies described herein can be expressed, for example, in a cell-free environment in vitro. The synthesis of two antibody half molecules with different specificities either in a single molecule or using co-expression The heavy chain CH3 interface in each half molecule is placed to favor the formation of heterodimers. By introducing the Fab arm exchange (or half-exchange) between two monospecific bivalent antibodies, Fab arm exchange reactions can be generated using disulfide bond isomerization. This is the result of the reaction and dissociation-association of the CH3 domain. The resulting free disulfide bonds of one of the parent monospecific antibodies are reduced. The antibody binds to the cysteine ​​residues of the second parent monospecific antibody molecule via intra-heavy chain disulfide bonds. At the same time, the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the ab arm appears to favor heterodimerization over homodimerization. The resulting products may each be engineered to target a different epitope, i.e., CD3 two Fab arms or halves that bind to an epitope on one antigen and an epitope on a second antigen It is a bispecific antibody having a

[0112] "Homodimerization" refers to the interaction of two heavy chains with the same CH3 amino acid sequence. "Homodimer" means a dimer having two heavy chains with identical CH3 amino acid sequences. It means an antibody that

[0113] "Heterodimerization" refers to the mutual dimerization of two heavy chains having non-identical CH3 amino acid sequences. "Heterodimer" refers to a heterodimer consisting of two dimers with non-identical CH3 amino acid sequences. The term "antibody" refers to an antibody having a heavy chain of

[0114] In some embodiments, the bispecific antibody is Triomab / Quadroma (T rion Pharma / Fresenius Biotech), Knob-in-H ole (Genentech), CrossMAb (Roche), and electrostatic match (C hugai, Amgen, NovoNordisk, Oncomed), LUZ-Y(G enentech), Strand Exchange Engineered Dom ain body(SEEDbody)(EMD Serono), Biclonic( Merus), and DuoBody® Technology (Genmab This includes design such as A / S.

[0115] Using triomab quadroma technology, VH and VH of the anti-CD3 antibody of the present invention were isolated. It is possible to generate full-length bispecific antibodies incorporating VLs. Triomab technology The study was carried out using two parent chimeric antibodies, one parent mAb with IgG2a and one parent mAb with rat IgG2b constant Fab arm exchange between the second parent mAb and the chimeric bispecific antibody results.

[0116] "Knobs-in-holes" strategies (see, e.g., WO 2006 / 028936) (preferably 100-2000) can be used to generate full-length bispecific antibodies. The selected amino acids that form the interface of the CH3 domain in human IgG are heterodimers. To promote mer formation, the ribosomal domains were mutated at positions that affect CH3 domain interactions. The amino acid with a small side chain (hole) can be used to generate an antibody that specifically binds to the first antigen. The heavy chain of the antibody is introduced with an amino acid (knob) with a large side chain that specifically binds to the second antigen. After co-expression of the two antibodies, the heterodimer is called a "Holstein-type" antibody. It is formed as a result of the preferential interaction of heavy chains with "knobs" and heavy chains with "holes." Exemplary CH3 substitution pairs that form knobs and holes are T366Y / F405A, T3 66W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T3 66S_L368A_Y407V (modification in the first CH3 domain of the first heavy chain) (expressed as modification position / modification position in the second CH3 domain of the second heavy chain).

[0117] CrossMAb technology can be used to generate full-length bispecific antibodies. rossMAb utilizes a "knob-in-hole" strategy to promote Fab arm exchange In addition, one of the half arms is required to ensure the correct light chain pairing of the resulting bispecific antibody. The CH1 and CL domains have been swapped to ensure (See No. 42,247).

[0118] Other crossover strategies can be used to crossover heavy chains in either one arm or both arms. Between the light chain or within the heavy chain of a bispecific antibody, the variable region or constant region, or both By exchanging domains, full-length bispecific antibodies can be generated. Examples of such exchanges include those described in International Publication Nos. 2009 / 080254 and 2009 / 0802 51, 2009 / 018386, and 2009 / 080252. Examples include VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1. can be.

[0119] Other strategies, such as combining positively charged residues on one CH3 surface and Heavy chain heterodimers using electrostatic interactions by replacing negatively charged residues in The promotion of merization is described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. It can be used as described in patent application publication no. 2011 / 0123532. In another strategy, heterodimerization is performed as described in U.S. Patent Application Publication No. 2012 / 0149876 or is substituted with the following substitutions: L3 as described in U.S. Patent Application Publication No. 2013 / 0195849 51Y_F405A Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407 V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T3 66V_K409F, Y407A / T366A_K409F, or T350V_L351 Y_F405A_Y407V / T350V_T366L_K392L_T394W (1st Modification position in the first CH3 domain of the first heavy chain / in the second CH3 domain of the second heavy chain This may be facilitated by (expressed as the modification position in

[0120] LUZ-Y technology can be used to generate bispecific antibodies. Wranik et al.,(2012)J Biol Chem 287(52): A leucine zipper was added to the C-terminus of the CH3 domain as described in 42221-9. It drives heterodimer assembly from the parent mAb, which is removed after purification.

[0121] SEEDbody technology can be used to generate bispecific antibodies. The body, in their constant domain, is 70, substituted with IgA residues to promote heterodimerization. With select IgG residues.

[0122] The bispecific antibodies described herein may be any of the antibodies described in WO 2011 / 131746. According to the method, asymmetric mutations were introduced into the CH3 regions of two monospecific homodimeric antibodies. , two parent monospecific homodimeric antibodies under reducing conditions that cause disulfide bonds to isomerize. By forming bispecific heterodimeric antibodies from In this method, a first monospecific bivalent antibody (i.e., a second antigen) can be produced in vitro. an antibody that specifically binds to, for example, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-I L1RAP antibody) and a second monospecific bivalent antibody (e.g., anti-CD3 antibody) engineered to have specific substitutions in the CH3 domain that promote immersion stability However, these antibodies have a cysteine ​​in the hinge region that isomerizes disulfide bonds. are incubated together under reducing conditions sufficient to induce Fab arm exchange. The bispecific antibody is generated by the reaction. The incubation conditions are then returned to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol, Dithioerythritol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxylate) carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol For example, at a temperature of at least 20°C and in the presence of at least 25 mM 2-MEA. in the presence of 0.5 mM dithiothreitol or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, e.g., Incubation at pH 7.0 or 7.4 for at least 90 minutes can be used. .

[0123] In some embodiments described herein, the first domain specifically binds to CD3. and a second domain that specifically binds to a second antigen, 3. Contains at least one substitution in the constant domain.

[0124] In some embodiments described herein, at least one amino acid sequence in the CH3 constant domain of the antibody is At least one substitution is a K409R, a F405L, or a F405L and a R409K substitution. , residue numbering follows the EU index.

[0125] Antibody domains and numbering are well known. "Asymmetric" refers to the division of two distinct heavy chains within an antibody. This refers to non-identical substitutions in the two CH3 domains. The IgG1 CH3 region is usually , consisting of residues 341 to 446 on IgG1 (residue numbering according to the EU index).

[0126] In some embodiments described herein, the bispecific antibody is an IgG1 isotype and the F405L substitution in the first heavy chain (HC1) of the antibody compared to wild-type IgG1. This includes a K409R substitution in the second heavy chain (HC2) of the antibody.

[0127] In some embodiments described herein, the bispecific antibody is an IgG1 isotype and a K409R substitution in the first heavy chain (HC1) of the antibody compared to wild-type IgG1. The antibody contains a F405L substitution in the second heavy chain (HC2) of the antibody.

[0128] In some embodiments described herein, the bispecific antibody is an IgG4 isotype and compared to wild-type IgG4, the S228P substitution in HC1 and the S228P substitution in HC2 It contains the S228P, F405L, and R409K substitutions.

[0129] In some embodiments described herein, the bispecific antibody is an IgG4 isotype and S228P, F405L, and R4 in HC1 compared to wild-type IgG4. It contains a 09K substitution, as well as a S228P substitution in HC2.

[0130] In some embodiments described herein, the bispecific antibody is an IgG4 isotype and S228P, F234A in HC1, and L2 35A substitution, as well as S228P, F234A, L235A, F405L in HC2; and R409K substitution.

[0131] In some embodiments described herein, the bispecific antibody is an IgG4 isotype and S228P, F234A, and L235 in HC1 compared to wild-type IgG4. A, F405L, and R409K substitutions, and S228P, F234A in HC2; and containing the L235A substitution.

[0132] In some embodiments described herein, the bispecific antibodies of the invention have a nucleotide sequence at residue position 35 0, 366, 368, 370, 399, 405, 407, or 409 in HC1 and At least 1, 2, 3, 4, 5, 6, 7, or 8 mismatches in HC2 The residue numbering is according to the EU index.

[0133] In some embodiments described herein, the bispecific antibodies of the invention have a nucleotide sequence at residue position 35 0, 370, 405, or 409 in HC1 and HC2, at least one, 2 The amino acid sequence contains one, three, or four asymmetric substitutions, and residue numbering is according to the EU index.

[0134] In some embodiments described herein, the bispecific antibodies of the invention have a nucleotide sequence at residue position 40 5 or 409, containing at least one asymmetric substitution in HC1 and HC2, The numbering of the bases follows the EU index.

[0135] Substitutions are typically made using standard methods to DNA fragments of molecules such as the constant domain of an antibody. It is done at A level.

[0136] The antibodies of the present invention may be modified into a variety of well-known antibody forms.

[0137] In some embodiments, the bispecific antibodies of the invention are crossbodies.

[0138] In some embodiments, the bispecific antibodies of the invention include recombinant IgG-like dual-target molecule (each of the two sides of the molecule is a Fab fragment or Fab fragment of at least two different antibodies) IgG fusion molecules (full-length IgG antibodies containing extra Fab or Fa fragments) b fragment), Fc fusion molecules (single chain Fv molecules or stabilized bispecific Fab fusion molecules, in which a specific antibody is fused to a heavy chain constant domain, Fc region, or portion thereof (different Fab fragments are fused together), ScFv and diabody-based antibodies and Heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single chain Fv molecules or different double chain Fv molecules) Specific antibodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) can be fused to each other. or fused to another protein or carrier molecule).

[0139] In some embodiments, the recombinant IgG-like dual targeting molecule includes Dual Tar getting(DT)-Ig(GSK / Domantis), Two-in-one A antibody (Genentech), and mAb2 (F-Star).

[0140] In some embodiments, the IgG fusion molecule comprises a Dual Variable D omain(DVD)-Ig(Abbott), Ts2Ab(MedImmune / AZ ), BsAb (Zymogenetics), HERCULES (Biogen I dec), and TvAb (Roche).

[0141] In some embodiments, Fc fusion molecules include ScFv / Fc Fusions ( Academic Institution), SCORPION (Emergent BioSolutions / Trubion,Zymogenetics / BMS), and and Dual Affinity Retargeting Technology (Fc -DART) (MacroGenics).

[0142] In some embodiments, the Fab fusion bispecific antibody includes F(ab)2 (Med arex / AMGEN), Dual-Action or Bis-Fab(Genen tech), Dock-and-Lock (DNL) (ImmunoMedics), B ivalent Bispecific (Biotecnol), and Fab-Fv (U CB-Celltech). ScFv-, diabody-based antibodies and dopamine- The main antibody is Bispecific T Cell Engager (BITE )(Micromet), Tandem Diabody(Tandab)(Affim ed), Dual Affinity Retargeting Technology (DART)(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, Recep torLogics), Human Serum Albumin ScFv Fusi on (Merrimack), and COMBODY (Epigen Biotech), Dual targeting nanobody (Ablynx), dual targeting heavy chain only Examples include dual targeting heavy chain only domain antibodies. Bispecific antibodies in various formats have been described, for example, by Chames and Baty (2 009)Curr Opin Drug Disc Dev 12:276 and Nune z-Prado et al.,(2015)Drug Discovery Toda y 20(5):588-594.

[0143] Table 2 lists the antibodies described herein that can be used to generate bispecific antibodies of the invention. 1 summarizes exemplary monoclonal antibodies described.

[0144] [Table 2]

[0145] Examples that can be used to engineer bispecific TMEFF2 x CD3 molecules Exemplary anti-TMEFF2 antibodies are those described herein, including those listed in Table 59, Heavy chains, including but not limited to the heavy and light chain variable region sequences provided in Tables 66-68 and a light chain variable region sequence.

[0146] In some embodiments, the anti-CD3 antibody is a multispecific antibody, e.g., a CD3 specific antibody. and a second domain that specifically binds to a second antigen. In some embodiments, the second or target antigen is an immune effector In some embodiments, the second antigen is a cell surface antigen expressed on a target cell other than the target cell. The antigen is a TAA. Exemplary TAAs include PSMA, CD33, TMEFF2, and I It's L1RAP.

[0147] The present invention relates to a method for detecting a CD3-specific antigen comprising administering to a subject a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. and a second domain comprising:

[0148] In some embodiments, the first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 666, HCDR1, HCDR2, HCDR3, LCDR1 of 4, 671, 673, and 690, In some embodiments, the first domain comprises LCDR2, and LCDR3. In some embodiments, the first comprises the VH and VL of SEQ ID NOs: 652 and 661, respectively. The domains include the HC and LC of SEQ ID NOs: 640 and 676, respectively. In one embodiment, the first domain comprises a VH and a VL of SEQ ID NOs: 657 and 678, respectively. In some embodiments, the first domain comprises the H of SEQ ID NOs: 675 and 677, respectively. Includes C and LC.

[0149] In some embodiments, the first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 666, HCDR1, HCDR2, HCDR3, LCDR1 of 4, 773, 673, and 690, In some embodiments, the first domain comprises LCDR2, and LCDR3. In some embodiments, the first comprises a VH and a VL of SEQ ID NOs: 657 and 678, respectively. The domains include the HC and LC of SEQ ID NOs: 675 and 677, respectively.

[0150] The present invention also provides a method for treating a subject with a leukemia virus that specifically binds to CD3 of the present invention. a bispecific antibody comprising one domain that specifically binds to a second antigen and a second domain that specifically binds to a second antigen; to provide.

[0151] The present invention also provides a method for treating a cell proliferative disorder, comprising administering to a subject a CD3-specific antibody of the present invention. a first domain that specifically binds to a second antigen and a second domain that specifically binds to a second antigen. , bispecific antibodies are provided.

[0152] The present invention also provides antibodies that specifically bind to CD3 of the present invention for use in the treatment of cancer. and a second domain that specifically binds to a second antigen. An antibody is provided.

[0153] The present invention also provides a CD3-specific antibody of the invention for use in the treatment of autoimmune diseases. a first domain that specifically binds to a second antigen and a second domain that specifically binds to a second antigen, Bispecific antibodies are provided.

[0154] The present invention also relates to a compound of the present invention, comprising a compound of the present invention, ... A first domain that specifically binds to D3 and a second domain that specifically binds to a second antigen and

[0155] The present invention also provides a compound comprising: The present invention relates to a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. and a domain of

[0156] A further aspect of the present invention is to provide a method for treating a cell proliferative disorder or autoimmune disease in a subject in need thereof. A method for treating an infectious disease comprising administering to said subject a therapeutically effective amount of an anti-CD3 antibody of the invention. In some embodiments, the method comprises administering an anti-CD3 antibody or a CD3 antibody of the present invention. a first domain that specifically binds to antigen 3 and a second domain that specifically binds to a second antigen; The bispecific antibody comprising the compound is administered to a subject at a dose of about 0.01 mg / kg to about 10 mg / kg. In some embodiments, an anti-CD3 antibody or antibody specifically binding to CD3 of the present invention is administered. and a second domain that specifically binds to a second antigen. The antibody is administered to the subject at a dose of about 0.1 mg / kg to about 10 mg / kg. In this embodiment, the anti-CD3 antibody or first domain that specifically binds to CD3 of the present invention and a second domain that specifically binds to a second antigen, In some embodiments, the anti-CD3 antibody or the antibody of the present invention is administered to a subject at a dose of 1 kg. The antibody comprises a first domain that specifically binds to CD3 of a target antigen and a second domain that specifically binds to a second antigen. Bispecific antibodies containing insulin can be administered subcutaneously, intravenously, intramuscularly, topically, orally, orally. skin, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or In some embodiments, the anti-CD3 antibodies, or CD3-specific antibodies of the present invention, are administered intranasally. a first domain that specifically binds to a second antigen and a second domain that specifically binds to a second antigen. The bispecific antibody is administered subcutaneously. In some embodiments, the anti-CD3 antibody or the A first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. The bispecific antibody comprising the main antibody is administered intravenously.

[0157] In any of the foregoing uses or methods, the cell proliferative disorder is cancer. In terms of form, cancers include esophageal cancer, gastric cancer, small intestine cancer, colon cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-small cell lung cancer, Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, renal cell carcinoma, Prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B cell-like (G CB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), Myeloid cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) LL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LPL) Lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system phospholipid CNSL, Burkitt's lymphoma (BL), B-cell lymphocytic leukemia, splenic marginal zone leukemia Lymphoma, hairy cell leukemia, splenic lymphoma / leukemia unclassifiable, diffuse red spleen Small medullary B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's macroglobulin Globulinemia, heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma , Pediatric nodal marginal zone lymphoma, Pediatric follicular lymphoma, Primary cutaneous follicle center lymphoma, T cell / Histiocytic-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLB CL-lower limb type, EBV-positive DLBCL in elderly patients, DLBCL associated with chronic inflammation, lymphoid Granulomatous granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-associated multicentric lymphoma Large B-cell lymphoma, primary effusion lymphoma arising in Castleman's disease: Diffuse Unclassifiable B-cells with characteristics intermediate between idiopathic large B-cell lymphoma and Burkitt's lymphoma lymphoma, classical Hodgkin's lymphoma, and light chain amyloidosis. do.

[0158] In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is adenocarcinoma. For example, metastatic adenocarcinoma (eg, colorectal adenocarcinoma, gastric adenocarcinoma, or pancreatic adenocarcinoma).

[0159] In any of the foregoing uses or methods, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, juvenile arthritis, and the like. Rheumatism, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic hematologic malignancies Thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia IgA nephropathy, IgM polyneuropathy, myasthenia gravis, hematopoietic disorders, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, hematopoietic disorders vasculitis, diabetes mellitus, Raynaud's syndrome, Sjögren's syndrome, glomerulonephritis, neuromyelitis optica ( NMO), and IgG neuropathy.

[0160] In another aspect, the present invention provides a method for the preparation of an antibody comprising: (a) a first domain that specifically binds to CD3 of the present invention; and a second domain that specifically binds to a second antigen. and (b) a composition comprising any one of the isomeric and isomeric antibodies, and a compound that is capable of treating or slowing the progression of a cell proliferative disorder. and a package insert containing instructions for administering the composition to a subject to achieve the desired effect. The term "package insert" refers to information about the indications, usage, dosage, and administration of such therapeutic products. , marketing notices for therapeutic products, including information on concomitant medications, contraindications, and / or warnings regarding use It is used to refer to the instructions normally included in the packaging of

[0161] In any of the above uses or methods, the subject may be a human.

[0162] Polynucleotides, Vectors, and Host Cells The anti-CD3 antibody of the present invention or the first domain of the present invention that specifically binds to CD3 and a second domain that specifically binds to a second antigen. Also disclosed are isolated polynucleotides capable of encoding the variable domains provided herein. The isolated polynucleotides capable of being isolated may be used in the same or similar manner to produce antibodies or antigen-binding fragments of the invention. may be contained in different vectors.

[0163] In some embodiments, the polynucleotide of the invention encodes a leader sequence. Any leader sequence known in the art may be used. The polynucleotide encoding the leader sequence can be It may contain a DNA cleavage site or a translation initiation site.

[0164] Also provided are vectors comprising the polynucleotides of the invention. The vectors are expression vectors. The expression vector may contain one or more additional sequences, including: For example, regulatory sequences (e.g., promoters, enhancers), selectable markers, and polyadenylation sequences. These include, but are not limited to, ylation signals. Vectors for this purpose are well known and include plasmids, phagemids, cosmids, baculoviruses, and the like. DNA fragments, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other These include, but are not limited to, bacterial, yeast, and viral vectors.

[0165] Recombinant expression vectors within the scope of this specification are operably linked to suitable control elements. a synthetic or cDNA encoding at least one recombinant protein capable of ligating Such regulatory elements include transcription promoters, suitable nucleic acid fragments derived from A. Sequences encoding mRNA ribosome binding sites and sequences controlling the termination of transcription and translation Expression vectors, particularly mammalian expression vectors, can also contain one or more non-transcriptional elements, such as an origin of replication, a suitable promoter linked to the gene to be expressed, promoters and enhancers, other 5' or 3' flanking non-transcribed sequences, 5' or 3' untranslated sequences sequences (e.g., essential ribosome binding sites), polyadenylation sites, splice donors and It may contain an acceptor site, or a transcription termination sequence. An origin of replication for the transcription factor may also be included.

[0166] The transcriptional and translational control sequences in expression vectors used to transform vertebrate cells are , and viral resources. An exemplary vector is Okayama a and Berg, 3 Mol. Cell. Biol. 280 (1983) It may be constructed as follows. Biol. 280 (1983).

[0167] In some embodiments, the coding sequence for the antibody or antigen-binding fragment comprises a strong construct. promoters, such as those for the gene: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human placed under the control of promoters for osin, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for the described implementation. Such viral promoters are suitable for use in the form of cytomegalovirus. CMV intermediate-early promoter, SV40 early and late promoters, mouse mammary tumor virus Virus (MMTV) promoter, Moloney leukemia virus, human immunodeficiency virus (HI V), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and other Retroviral long terminal repeats (LTRs) and herpes simplex virus thymidine In one embodiment, the promoters include, but are not limited to, PSMA promoters. The coding sequence for the heterologous antibody or antigen-binding fragment thereof may be driven by an inducible promoter, e.g., a metallothio Nein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter data, one or more interferon stimulated response elements (ISREs), e.g., protein kinase R2',5'-oligoadenylate synthetase, Mx gene, ADAR1, etc. It is placed under the control of a promoter containing

[0168] The vectors described herein may contain one or more internal ribosome entry sites (IRES). Inclusion of an IRES sequence in a fusion vector may facilitate the expression of some proteins. In some embodiments, the vector system may comprise one or two The polyadenylation site will contain one or more polyadenylation sites (e.g., SV40). The components of the vector may be adjacent to one another or may be located upstream or downstream of one of the nucleic acid sequences. The genes may be linked at intervals (all at once) to provide optimal spacing for expression of the gene product. (i.e., by introducing "spacer" nucleotides between ORFs) Regulatory elements, such as IRES motifs, may be located within or otherwise within the The probes may also be spaced to provide optimal spacing for expression.

[0169] The vector may contain a selectable marker known in the art. Examples include positive and negative selection markers, such as antibiotic resistance genes (e.g., neomycin). cytosine resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetrasaccharin resistance gene Icrin resistance gene, penicillin resistance gene), glutamate synthase gene, cancer HSV-TK, HSV-TK derivatives for cyclovir selection, or 6-methylpurine for selection Bacterial purine nucleoside phosphorylase genes (Gadi et al., 2007 Gen e Ther.1738-1743(2000)). (Margin) Selection marker The nucleic acid sequence encoding the polypeptide of interest or the cloning site is It may be upstream or downstream of the nucleic acid sequence or cloning site.

[0170] The vectors described herein can be used to produce the described antibodies or antigen-binding fragments. A variety of cells can be transformed with genes encoding the vectors. , anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibody, or to generate cells that produce antigen-binding fragments. Also provided is a host cell comprising a vector of the invention.

[0171] Numerous techniques for introducing foreign genes into cells are known in the art. , for the purpose of carrying out the described methods, according to the various embodiments described and illustrated herein. The techniques used can be used to create recombinant cells in which the heterologous gene sequence is that are inheritable and expressible by the cell's progeny and that confer essential growth and physiological functions on the recipient cell The heterologous gene sequence is stably introduced into the host cell without impairing its biological function. The methods that can be used include chromosome transfer (e.g., cell fusion) , chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., Transfection, spheroplast fusion, microinjection, electroporation lation, liposome carriers), viral vector transduction (e.g., recombinant DNA viruses) , recombinant RNA viruses) (Cline, 29 Pharmac. The r.69-92(1985)) but are not limited to these. (1985)). Calcium phosphate precipitation of bacterial protoplasts and poly(A)-induced precipitation of bacterial protoplasts by mammalian cells. Ethylene glycol (PEG) induced fusion can also be used to transform cells. Cut.

[0172] Cells suitable for use in expressing the antibodies or antigen-binding fragments described herein are preferably is a eukaryotic cell, more preferably a cell of plant, rodent, or human origin, e.g. In particular, NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, and HEK29 3 cells, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, He These include, but are not limited to, La, NS1, Sp2 / 0 myeloma cells, and BHK cell lines. In addition, expression of antibodies may be achieved using hybridoma cells. Methods for producing domars are well established in the art.

[0173] Cells transformed with the expression vector of the present invention are used to express the recombinant antibody or antigen-binding fragment of the present invention. Recombinant positive cells may be selected or screened for expression of the protein modification or The altered post-translational modifications can result in desirable phenotypes, e.g., high levels of expression, improved growth, and Subclones that exhibit the desired properties or ability to produce proteins with the desired biochemical properties are selected. These phenotypes are used to characterize the identity of a given subclone. Mutations can be due to chemicals, UV light, radiation, viruses, or other factors. by cloning, insertional mutagenesis, inhibition of DNA mismatch repair, or a combination of these methods. This can occur.

[0174] Pharmaceutical Composition / Administration The present invention also provides a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the present invention are administered in an effective amount as the active ingredient in a pharmaceutically acceptable carrier. The antibody of the present invention can be prepared as a pharmaceutical composition containing the antibody. It refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such vehicles include: Water and oils, including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil The liquid may be a liquid such as 0.4% saline and 0.3% glycerin. These solutions are sterile and generally free of particulate matter. Sterilization may be achieved by conventional, well-known sterilization techniques (e.g., filtration). pharmaceutically acceptable auxiliary substances required to approximate environmental conditions, e.g., pH adjusting agents and buffering agents, stabilizers, thickening agents, lubricants, coloring agents, and the like. The concentration of the antibody of the present invention in the solution ranges from less than about 0.5% by weight to usually at least about 1% by weight, and most preferably from about 0.5% by weight to at least about 1% by weight. The amount may vary up to 15 or 20% by weight and may be adjusted as needed according to the particular method of administration selected. The vehicle may be selected primarily based on the required dose, fluid volume, viscosity, etc. Formulations (including other human proteins, e.g., human serum albumin) are available from, e.g., Remin gton:The Science and Practice of Pharmac y,21stEdition,Troy,DBed.,Lipincott Will liams and Wilkins,Philadelphia,PA 2006,P art 5,Pharmaceutical Manufacturing pp691 -1092, see especially pp. 958-989.

[0175] The administration method for therapeutic use of the antibody of the present invention can be, for example, a tablet, capsule, liquid, or the like. , powder, gel, particle, syringe, implantable device, osmotic pump, cartridge, microphone Parenteral administration using formulations contained in a pump, e.g., intradermal, intramuscular, intraperitoneal, intravenous intracutaneous, subcutaneous, intrapulmonary, transmucosal (oral, intranasal, intravaginal, rectal) administration; or Site-specific administration can be achieved by other means well known in the art and recognized by those skilled in the art. , intratumoral, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intrasinus, intracavity, intracerebellar, intraventricular, Intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, prostatic Intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal cord, intrasynovial bursa, intrathoracic, intrauterine, intravascular, bladder This may be achieved by intravenous, intralesional, intravaginal, intrarectal, buccal, sublingual, intranasal, or transdermal delivery. .

[0176] The antibodies of the invention can be administered by any suitable route, for example, by intravenous (iv) infusion or is administered to a subject parenterally, intramuscularly, subcutaneously, or intraperitoneally by bolus injection The iv infusion may be, for example, 15, 30, 60, 90, 120, 180 or 240 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours It can be administered over a period of time.

[0177] The dose given to the subject will alleviate or at least partially arrest the disease being treated. A therapeutically effective amount sufficient to induce a steroid effect (a "therapeutically effective amount"), sometimes between 0.005 mg and about 100 mg / kg For example, about 0.05 mg to about 30 mg / kg, or about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg, or about 24 mg / kg; Or it may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg However, even higher amounts, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 2 at 3, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg It's okay to have it.

[0178] For example, a fixed unit dose of 50, 100, 200, 500, or 1000 mg may be given. Or the dose may be calculated based on the surface area of ​​the patient, e.g., 500, 400, 300, 250, 200, or 100 mg / m 2 Usually, 1 to 8 doses (e.g. , 1, 2, 3, 4, 5, 6, 7, or 8) are administered to treat the patient, but 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more A lower dose may be given.

[0179] The antibody of the present invention may be administered on the following days: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks After 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, The course of treatment may be repeated or extended over a longer period. Repeated administrations may be of the same or different doses. For example, the antibodies of the invention described herein can be administered by intravenous infusion over an 8 week period. The patients were administered 8 mg / kg or 16 mg / kg weekly, followed by a further 16 weeks of The patients were administered 8 mg / kg or 16 mg / kg every 2 weeks, followed by 8 mg / kg every 4 weeks. It may be administered at 16 mg / kg or 16 mg / kg.

[0180] For example, the antibodies in the methods described herein may be administered in a single dose or in 24, 12, 8, 6, about 0.1, 4, or 2 hourly divided doses, or any combination thereof ~100mg / kg, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 2 per day , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 , as a daily dosage in amounts of 45, 50, 60, 70, 80, 90, or 100 mg / kg , after starting treatment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th or 40th day At least one of the days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, At least one of the 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th weeks or any combination thereof.

[0181] The antibodies described herein may also be used to treat cancer progression, reduce the risk of developing cancer, delay the onset of events in the patient and / or reduce the risk of recurrence when the cancer goes into remission It may also be administered prophylactically to reduce

[0182] The antibodies provided herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations. Well known lyophilization and reconstitution techniques can be used.

[0183] CD3, PSMA, CD33, IL1RAP, TMEFF2, or target antigen and CD3 How to detect Detect CD3, PSMA, CD33, TMEFF2, or IL1RAP in a sample The method includes obtaining a sample, and treating the sample with an anti-CD3 antibody, an anti-PSMA antibody, or an antibody of the present invention. contacting the sample with an anti-CD33, anti-TMEFF2, or anti-IL1RAP antibody; Antibodies bound to CD3, PSMA, CD33, TMEFF2, or IL1RAP in the pool and detecting the

[0184] Additionally, CD3 and a second antigen (e.g., PSMA, CD33, TMEFF) in the sample may be detected. 2, or IL1RAP), the method comprising obtaining a sample; a first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen, contacting the sample with a bispecific antibody containing the domain; and detecting CD3 and the target domain in the sample. and detecting the antibody bound to the two antigens.

[0185] In some embodiments described herein, the sample may be urine, blood, serum, plasma, saliva, or the like. , ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue tissues (e.g., surgically resected tumor tissue, biopsies including fine needle aspiration), histological preparations, etc. It may be derived from.

[0186] Antibodies of the present invention can be detected using known methods. Exemplary methods include directly labeling the antibody with a fluorescent or chemiluminescent label, or a radiolabel; Alternatively, the antibodies of the invention may be tagged with an easily detectable moiety such as biotin, an enzyme, or an epitope tag. Exemplary labels and moieties include ruthenium, 111 In-D OTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxide oxidase, alkaline phosphatase and beta-galactosidase, poly-histidine ( HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthroline dyes, Nanthridine dyes, rhodamine dyes, and Alexafluor® dyes .

[0187] The antibodies provided herein are capable of detecting CD3, PSMA, CD33, IL1RA in a sample. Used in various assays to detect P, TMEFF2, or CD3, and a second antigen. Exemplary assays include Western blot analysis, radioimmunoassay, and the like. Sequencing, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA It is an assay.

[0188] Antibody Kits The present invention also relates to an antibody against anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL-1. 1RAP antibody, or a first domain that specifically binds to CD3 of the present invention and a second antigen or a bispecific antibody comprising a second domain that specifically binds to the antibody or an antigen-binding fragment thereof; Kits containing one or more of these antigen-binding fragments are also provided. , anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibody, Alternatively, the present invention relates to a first domain that specifically binds to CD3 and a second domain that specifically binds to an antigen. or other methods known to those skilled in the art. In some embodiments, the kits described can be used to perform the methods. The present invention relates to a method for detecting CD3 or PSMA in a biological sample, comprising administering to a subject an antibody or antigen-binding fragment described herein. , CD33, IL1RAP, TMEFF2, or other antigens. The kits described may therefore comprise the components described herein. an antibody or antigen-binding fragment thereof, and a container for housing the antibody or fragment when not in use; a container for storing the antibody or fragment; instructions for use of the antibody or fragment; an antibody or fragment immobilized on a solid support; fragments, and / or detectably labeled forms of the antibodies or fragments described herein. It may include one or more of the following:

[0189] The present invention also provides kits that include the antibodies described herein that specifically bind to PSMA. The present invention also provides an antibody described herein that specifically binds to CD33. Kits are also provided. The present invention also provides a method for detecting IL1RAP using a method described herein that specifically binds to IL1RAP. The present invention also provides a kit containing the antibody. Kits are also provided that include the antibodies described herein.

[0190] The kits can be used for therapeutic use and as diagnostic kits.

[0191] The kit detects CD3, PSMA, CD33, IL1RAP, and TMEFF in biological samples. 2, and / or may be used to detect the presence of a second antigen.

[0192] In some embodiments, the kit comprises an antibody of the invention described herein and a method for detecting the antibody. The kit may also include instructions for use; other reagents, such as labels, therapeutic agents, or to an agent, label or therapeutic agent useful for chelation or otherwise coupling or radioprotective compositions; devices or other means for preparing such antibodies for administration; Materials; including pharmaceutically acceptable carriers, and devices or other materials for administration to a subject , which may include one or more other components.

[0193] In some embodiments, the kit comprises an antibody of the invention in a container and instructions for use of the kit. Includes.

[0194] In some embodiments, the antibody in the kit is labeled.

[0195] CD3 specific antibody Isolated anti-CD3 antibodies are described herein. The anti-CD3 antibodies of the present invention are isolated from human CD3 and Optionally, the anti-CD3 of the invention binds to cynomolgus monkey CD3. The antibody and its fragments bind to human and cynomolgus CD3 with affinities within 5-fold of each other In other words, the difference in antibody binding is less than 5-fold. In this case, the anti-CD3 antibodies of the present invention Preclinical evaluation of the safety, activity, and / or pharmacokinetic profile of CD3 in primates In other words, the same CD3 The specific molecules can be used in preclinical animal studies as well as in clinical trials in humans. This human / cynomolgus cross-reactivity is highly comparable compared to species-specific surrogate molecules. In some embodiments, the present invention provides a method for the preparation of a medicament for the treatment of cancer, resulting in a significant increase in the predictive power of the results and animal studies. Anti-CD3 antibodies and their fragments target epitopes formed by the CD3e / d subunits. CD3-specific antibodies may be human, humanized, or chimeric. Also, OmniRat (Open Monoclonal Technology) Logies (“OMT”), Palo Alto, California, USA, o Human antibodies generated at niab.com are also exemplified herein.

[0196] In some embodiments described herein, the anti-CD3 antibody or fragment thereof has the following characteristics: having one, two, three, four, or five of: a) Binds to human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 20 nM or less and binds to cynomolgus monkey CD3-expressing HEK cells with a calculated EC50 of 40 nM or less, and CD3 Calculated EC5 between binding to +T lymphocytes and binding to cynomolgus monkey CD3-expressing HEK cells The difference in EC50 values ​​is less than 5-fold. as measured by cytoplasmic binding assay; b) an equilibrium dissociation constant (K) of 12 nM or less D ) and human-derived recombinant CD3d (SEQ ID NO: 69 1), or binds to recombinant human CD3e (SEQ ID NO: 636), or binds to cynomolgus monkey It binds to recombinant CD3e (SEQ ID NO: 693) derived from K D Store Proteo at +25°C. n Surface plasmon resonance assay measured using the ProteOn XPR36 system ru; c) The oxidation of methionine and tryptophan as detected by peptide mapping analysis nor oxidation of asparagine, nor deamidation of asparagine, nor isomerization of asparagine. does not show; d) binds to residues 1-6 of CD3e as determined by X-ray crystallography; or e) cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay Activates T cells or induces CD69 expression to a similar extent.

[0197] The anti-CD3 antibodies and fragments thereof of the present invention have the following properties as determined by flow cytometry: About 5 nM to about 1000 nM, preferably about 5 nM to about 50 nM, about 50 nM to about 100 nM M, about 100nM to about 200nM, about 200nM to about 300nM, about 300nM to about 40 0nM, about 400nM to about 500nM, about 500nM to about 600nM, about 600nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, and about 900 The concentration of human CD3 is about 5 nM to about 1000 nM, more preferably about 5 nM to about 300 nM. In vitro binding affinity (K) for expressing human T cells d )

[0198] In some embodiments, the anti-CD3 antibodies and fragments thereof of the present invention are capable of detecting CD3 fragments by flow cytometry. When determined based on this, the concentration is about 5 nM to about 1000 nM, preferably about 5 nM to about 50 nM, and 50nM to about 100nM, about 100nM to about 200nM, about 200nM to about 300nM, Approx. 300nM ~ Approx. 400nM, Approx. 400nM ~ Approx. 500nM, Approx. 500nM ~ Approx. 600n M, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 90 0 nM, and about 900 nM to about 1000 nM, more preferably about 5 nM to about 300 nM, Most preferably, the antibody has an intensity of about 100 nM for human T cells expressing human CD3. In vitro binding affinity (K d ) is a bivalent antibody having

[0199] In some embodiments, the anti-CD3 antibodies and fragments thereof of the present invention are capable of detecting CD3 fragments by flow cytometry. When determined based on this, the concentration is about 5 nM to about 1000 nM, preferably about 5 nM to about 50 nM, and 50nM to about 100nM, about 100nM to about 200nM, about 200nM to about 300nM, Approx. 300nM ~ Approx. 400nM, Approx. 400nM ~ Approx. 500nM, Approx. 500nM ~ Approx. 600n M, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 90 0 nM, and about 900 nM to about 1000 nM, more preferably about 100 nM to about 250 nM M, most preferably about 250 nM, against human T cells expressing human CD3 In vitro binding affinity (K d ) is a monovalent construct having

[0200] In one aspect, the anti-CD3 antibodies and fragments thereof described herein are capable of detecting CD3 activity using flow cytometry. Measured on primary human T cells using AlexaFluor488 conjugates As determined by a competitive binding assay using the SP34-2 antibody, The commercially available CD3 antibody SP34-2 (BD Biosciences 551916) was used. Compete.

[0201] In one aspect, the anti-CD3 antibodies and fragments thereof have a , does not exhibit post-translational modifications, for example, does not exhibit oxidation, does not exhibit deamidation, and aspartic acid does not exhibit isomerization.

[0202] In one aspect, the anti-CD3 antibodies and fragments thereof are used in T cell-based immunoassays using flow cytometry. SP34-2 in human and cynomolgus monkey T cells as determined by the ELISA assay It also activates T cells to a similar extent as cOKT3 in human T cells, and expresses CD69. It is effective in inducing

[0203] In one aspect, the anti-CD3 antibodies and fragments thereof described herein have a vasoconstriction of about 400 kcal / mo l or more, about 410kcal / mol or more, about 420kcal / mol or more, about 430kc al / mol or more, about 440 kcal / mol or more, about 45 kcal / mol or more, about 4 60kcal / mol or more, about 470kcal / mol or more, about 480kcal / mol or more, about 490kcal / mol or more, about 500kcal / mol or more, about 510kca l / mol or more, about 520kcal / mol or more, about 530kcal / mol or more, about 5 Unfolding of more than 40 kcal / mol or about 550 kcal / mol In certain embodiments, the anti-CD3 antibodies and fragments thereof of the present invention have a total enthalpy of about 418 kcal / mol, 545 kcal / mol, approximately 402 kcal / mol, or approximately The total enthalpy of unfolding of the anti-CD3 antibody is 406 kcal / mol. are CD3B376 (IgG4 PAA) and CD3B450 (IgG4 PAAs), respectively. A), CD3B389 (IgG1σ), and CD3B467 (IgG1σ) molecules.

[0204] Exemplary such antibodies include CD3B311, CD3B312, CD3B313 , CD3B314, CD3B315, CD3B316, CD3B317, CD3B334 , CD3B376, CD3B389, CD3B450, as well as genes in monovalent formats Engineered CD3B467, CD3B376 and CD3B450 are included.

[0205] The anti-CD3 antibodies or antigen-binding fragments of the present invention can exist in a variety of forms, including the following: 7A and its genetically engineered antibody variable domain segments or CDRs. Variants, e.g., those shown or described in Tables 9 and 10 and the accompanying descriptions. It includes one or more of the following:

[0206] The present invention also provides the HCDR1, HCDR2, and HCDR3 sequences of any one of the antibodies listed in Table 7B. The present invention provides an anti-CD3 antibody or antigen-binding fragment thereof, comprising a heavy chain comprising an HCDR3. and the HCDR1, HCDR2, and HCDR3 of any one of the antibodies listed in Table 7B. and a heavy chain comprising the LCDR1, LCDR2, and LCDR3 of any one of the antibodies listed in Table 7B. and a light chain comprising DR3. In one embodiment, the anti-CD3 antibody or antigen-binding fragment thereof of the invention is an antibody of Table 7B. a heavy chain comprising any one of HCDR1, HCDR2, and HCDR3 and a heavy chain comprising any one of HCDR1, HCDR2, and HCDR3 as set forth in Table 7B a light chain comprising LCDR1, LCDR2, and LCDR3 of any one of antibodies The antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof for binding to CD3.

[0207] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises SEQ ID NO:6 51, 652, 653, 654, 655, 687, or 656 in the heavy chain variable region (VH) HCDR1, HCDR2, and HCDR3, DR2 and HCDR3 are defined by Chothia, Kabat, or IMGT. can be.

[0208] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises SEQ ID NO:6 58, 659, 694, 660, 688, or 661 LCDR1, LCDR2, and LCDR3, and LCDR3 as defined by Chothia, Kabat, or IMGT.

[0209] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises: : HCDR1 of SEQ ID NO: 662, 665, or 666; HCDR2 of SEQ ID NO: 663, 689, or 695; HCDR3 of sequence number 664.

[0210] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises: : LCDR1 of SEQ ID NO: 773, 710, 674 or 671; LCDR2 of SEQ ID NO: 669 or 673; LCDR3 of sequence number 670.

[0211] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises: : HCDR1 of SEQ ID NO: 662, 665, or 666; HCDR2 of SEQ ID NO: 663, 689, or 695; HCDR3 of SEQ ID NO: 664; LCDR1 of SEQ ID NO: 773, 710, 674 or 671; LCDR2 of SEQ ID NO: 669 or 673; LCDR3 of sequence number 670.

[0212] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention have the following HC Contains DR1, HCDR2, and HCDR3: SEQ ID NOs: 662, 663, and 664, respectively; SEQ ID NOs: 662, 695, and 664, respectively; SEQ ID NOs: 665, 663, and 664, respectively; SEQ ID NOs: 665, 695, and 664, respectively; SEQ ID NOs: 662, 689, and 664, respectively; SEQ ID NOs: 666, 663, and 664, respectively; SEQ ID NOs: 666, 695, and 664, respectively; SEQ ID NOs: 665, 689, and 664, respectively; or SEQ ID NOs: 666, 689, and 664, respectively.

[0213] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention comprise the following LC DR1, LCDR2, and LCDR3, including: SEQ ID NOs: 773, 669, and 670, respectively; SEQ ID NOs: 773, 673, and 670, respectively; SEQ ID NOs: 710, 673, and 670, respectively; SEQ ID NOs: 674, 673, and 670, respectively; SEQ ID NOs: 671, 673, and 690, respectively; SEQ ID NOs: 773, 673, and 690, respectively; SEQ ID NOs: 671, 669, and 670, respectively; or SEQ ID NOs: 776, 673, and 670, respectively.

[0214] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 773, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0215] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 773, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0216] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 671, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0217] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 671, 673, and 690 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0218] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 695, 664, 773, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0219] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 695, 664, 773, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0220] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 695, 664, 671, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0221] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 695, 664, 671, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0222] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 663, 664, 773, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0223] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 663, 664, 773, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0224] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 663, 664, 671, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0225] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 663, 664, 671, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0226] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 695, 664, 773, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0227] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 695, 664, 773, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0228] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 695, 664, 776, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0229] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 665, 695, 664, 776, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0230] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 663, 664, 773, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0231] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 663, 664, 773, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0232] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 663, 664, 776, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0233] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 663, 664, 671, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0234] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 695, 664, 773, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0235] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 695, 664, 773, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0236] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 695, 664, 671, 669, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0237] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 666, 695, 664, 671, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0238] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 689, 664, 671, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0239] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 710, 673, and 670 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0240] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 671, 673, and 690 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0241] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention each have the sequence HCDR1 and HCDR2 for column numbers 662, 663, 664, 773, 673, and 690 , HCDR3, LCDR1, LCDR2, and LCDR3.

[0242] In some embodiments, the antigen-binding fragment of an anti-CD3 antibody or antigen-binding fragment thereof of the invention heavy chain of SEQ ID NO: 709, 640, 641, 642, 643, 675, or 644 (HC) sequence, and / or SEQ ID NOs: 645, 716, 649, 676, 677, or In some embodiments, the antigen-binding fragments of the present invention comprise a light chain (LC) sequence of 650. The CD3 antibody or antigen-binding fragment thereof is selected from SEQ ID NOs: 709, 640, 641, 642, 643 , 675, or 644 and at least 85%, preferably 90%, more preferably 95% or more or more, e.g., 95%, 96%, 97%, 98%, or 99% identical polypeptide sequence. HC, and SEQ ID NO: 645, 716, 649, 676, 677, or 650 At least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% 98%, or 99% identical polypeptide sequence to the LC. In one embodiment, the antigen-binding fragment of the anti-CD3 antibody or antigen-binding fragment thereof of the present invention is SEQ ID NO: 709 , 640, 641, 642, 643, 675, or 644 and at least 85%, preferably is 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 9 HC, which has a polypeptide sequence 9% identical to SEQ ID NOs: 645, 716, 649, and 67 6, 677, or 650 and at least 85%, preferably 90%, more preferably 95% or more, e.g., 95%, 96%, 97%, 98%, or 99% identical polypeptide sequence. The LC contains the corresponding CDR sequences, and there is no sequence variation in the CDR regions.

[0243] In some embodiments, the antigen-binding fragment of an anti-CD3 antibody or antigen-binding fragment thereof of the invention is SEQ ID NO: 651, 652, 657, 653, 654, 655, 687, or 65 6 heavy chain variable region (VH) sequences, and / or SEQ ID NOs: 658, 659, 694, 660, 688, 678, or 661. In one embodiment, the antigen-binding fragment of the anti-CD3 antibody or antigen-binding fragment thereof of the present invention is SEQ ID NO: 651 , 652, 657, 653, 654, 655, 687, or 656 and at least 85%; Preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98% or a VH having a polypeptide sequence 99% identical thereto, and SEQ ID NOs: 658, 659, 69 4, 660, 688, 678, or 661 and at least 85%, preferably 90%, Preferably, the polynucleotide is 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the antigen-binding fragments of the invention comprise a VL having a peptide sequence. The CD3 antibody or antigen-binding fragment thereof is selected from SEQ ID NOs: 651, 652, 657, 653, 654 , 655, 687, or 656 and at least 85%, preferably 90%, more preferably A polypeptide sequence that is 95% or more, e.g., 95%, 96%, 97%, 98%, or 99% identical and a VH having the sequence SEQ ID NO: 658, 659, 694, 660, 688, 678, or 661 and at least 85%, preferably 90%, more preferably 95% or more, for example 95 96%, 97%, 98%, or 99% identical polypeptide sequence to the VL of There is no sequence variation in the CDR regions.

[0244] PSMA specific antibody Antibodies and fragments thereof that bind to PSMA bind to chimpanzee target antigens. In this embodiment, antibodies and fragments thereof are shown to bind human and macaque PSMA-targeted antibodies with affinities within 5-fold of each other. In other words, the difference in antibody binding is less than 5-fold. Preclinical studies of the safety, activity, and / or pharmacokinetic profile of PSMA in primates It can be used for evaluation and as a drug in humans. The PSMA-specific molecules will be used in preclinical animal studies as well as in human clinical trials. This allows for highly comparable results and predictability in animal studies compared to species-specific alternative molecules. The PSMA domain is species specific, i.e., human and mouse. Because the antibodies or fragments thereof are reactive with IgG antigens, they are able to inhibit the production of these antibodies in primates. For preclinical evaluation of the safety, activity, and / or pharmacokinetic profile of the combined domains; and can be used as a drug in humans in the same form.

[0245] The present invention also provides multispecific antibodies that specifically bind to PSMA. Therefore, bispecific, or bifunctional, antibodies can be used to engage two different therapeutic targets. Such antibodies can be used to detect and / or detect the presence of antibodies that bind to the target protein or that serve two different functions. Recruiting immune effector cells, such as T- or NK-cells, towards specific target cells A variety of antibody fragment-based molecules are known and are under investigation, for example, for cancer therapy. is.

[0246] The present invention also provides PSMA x "effector antigen" bispecific antibodies. In this form, the effector antigen of the PSMA x "effector antigen" bispecific antibody is CD The same molecule has been used in preclinical animal studies, as well as clinical trials and human treatments. It is now possible to generate PSMAxCD3 bispecific antibodies that can be used in This was found in the present invention to be due to the binding to human PSMA and human CD3, respectively. In addition, the PSMAxCD3 duplex also binds to homologs of chimpanzee and macaque antigens. The PSMAxCD3 bispecific antibody of the present invention is useful for the treatment of cancers including, but not limited to, cancers. In view of the above, it can be used as a therapeutic agent for various diseases, including but not limited to the following. , an alternative target PSMA x CD3 duplex for testing in phylogenetically distant species (from humans) As a result, the same molecule can be used in clinical trials in humans, eliminating the need to construct specific antibodies. It can be used in preclinical animal studies, such as those intended for administration to humans. In addition, it can be used in subsequent marketing approval and administration of therapeutic agents.

[0247] In some embodiments described herein, an isolated antibody or The antibody fragment has one, two, three, four, or five of the following properties: a. Equilibrium dissociation constant (K D ) and chimpanzee PSMA extracellular domain (E CD) and K D was measured using a ProteOn XPR36 system at +25°C. It is determined, b. Binds to LNCaP cells with a calculated EC50 of 20 nM or less and a calculated EC 50 binds to cynomolgus monkey PSMA-expressing HEK cells, and binds to LNCaP cells and cyno The difference in calculated EC50 between the binding of mAb to human PSMA-expressing HEK cells was less than 5-fold. Calculate EC50 measured in whole cell binding assay at 0 °C using flow cytometry will be c. Equilibrium dissociation constant (K) of 12 nM or less D ) and human (SEQ ID NO: 55), chimpanzee ( SEQ ID NO: 52), and recombinant PSMA ECD from cynomolgus monkey (SEQ ID NO: 53). S, K D Measure the surface plasmon resonance assay ProteOn X at +25°C. Measured using the PR36 system, d. When paired with anti-CD3 antibodies in a bispecific antibody, LNCaP cells, C4 2 cells, human PSMA-expressing HEK cells, or cynomolgus monkey PSMA-expressing HEK cells T cell-mediated killing, which is mediated by chromium-51 or caspase 3 as measured by the / 7 activation assay, or e. Recognizes a conformational epitope, which is a residue of human PSMA (SEQ ID NO: 51). Base I138, F235, P237, G238, D244, Y299, Y300, Q303 , K304, E307, and K324-P326

[0248] An exemplary such antibody or fragment thereof is the PSMA antibody PSMB11 described herein. 9, 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.

[0249] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 56, 57, 58, 59, 60, and 61, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0250] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 62, 63, 64, 65, 60, and 66, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0251] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 67, 68, 69, 70, 71, and 72, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0252] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 73, 74, 75, 76, 60, and 61, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0253] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 78, 79, 80, 81, 82, and 83, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0254] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 84, 85, 86, 87, 60, and 88, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0255] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0256] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 95, 96, 97, 65, 60, and 66, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0257] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have the HCDRs of SEQ ID NOs: 84, 98, 99, 100, 82, and 101, respectively. 1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0258] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies are those with HCs of SEQ ID NOs: 89, 90, 102, 103, 104, and 105, respectively. Includes DR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0259] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies are those with HCs of SEQ ID NOs: 89, 90, 106, 103, 104, and 105, respectively. Includes DR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0260] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies are HCDs of SEQ ID NOs: 107, 108, 109, 76, 60, and 88, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0261] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HDR2 and HDR3 of SEQ ID NOs: 78, 1, 80, 81, 82, and 83, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0262] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HDR2 and HDR3 of SEQ ID NOs: 78, 1, 80, 81, 82, and 83, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0263] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HDR2 and HDR3 of SEQ ID NOs: 78, 1, 80, 4, 82, and 686, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0264] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 of SEQ ID NOs: 78, 1, 80, 81, 792, and 686, respectively. , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0265] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HDR2 and HDR3 of SEQ ID NOs: 78, 2, 80, 81, 82, and 83, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0266] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 78, 3, 80, 81, 82, and 5, respectively. DR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0267] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HDR2 and HDR3 of SEQ ID NOs: 78, 3, 80, 81, 82, and 83, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0268] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HDR2 and HDR3 of SEQ ID NOs: 78, 3, 80, 4, 82, and 686, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0269] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 of SEQ ID NOs: 78, 3, 80, 81, 792, and 686, respectively. , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0270] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, HCDR7, HCDR8, HCDR9, HCDR10, HCDR11, HCDR12, HCDR13, HCDR14, HCDR15, HCDR16, HCDR17, HCDR18, HCDR19, HCDR1 ... DR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0271] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 78, 2, 80, 4, 792, and 686, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0272] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies have HCDR1 and HCDR2 of SEQ ID NOs: 78, 2, 80, 4, 792, and 686, respectively. Includes HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0273] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies are HCDs of SEQ ID NOs: 78, 683, 80, 81, 792, and 686, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0274] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. Antibodies that bind to the antibody sequences identified as SEQ ID NOs: 6, 7, 8, 110, 112, 114, 116, 118, 120, heavy chain variable region (VH) of 121, 123, 125, 126, 128, 130, or 681 In some embodiments of the invention described herein, the PSMA-specific antibodies of the invention include The antibodies that bind to SEQ ID NOs: 9, 111, 113, 115, 117, 119, 122, 1 24, 127, 129, 131, or 682 light chain variable regions (VL).

[0275] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies are represented by SEQ ID NOs: 12, 13, 132, 134, 136, 138, 140, 141, and heavy chain sequences of 143, 145, 146, 148, 150, 151, or 679.

[0276] In some embodiments of the invention described herein, the antibodies of the invention specifically bind to PSMA. The antibodies used are sequences 14, 15, 75, 133, 135, 137, 139, 142, and 1 The light chain sequences include 44, 147, 149, or 680.

[0277] CD33 specific antibody The CD33-specific antibodies of the present invention have high affinity binding to CD33 and / or CD3, High specificity for D33 and / or CD3, and efficacy alone or in combination with other anti-cancer therapies and the ability to treat or prevent cancer when administered in combination with one or more The above desirable functional properties are obtained.

[0278] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to the C of CD33. In certain embodiments, an isolated monoclonal antibody or its antigen-binding The fragment binds to the V domain of CD33. Full-length human CD33 is available from Uniprot P 20138 (SEQ ID NO: 244).

[0279] As used herein, an antibody that "specifically binds to CD33" refers to an antibody that specifically binds to CD33, preferably or 1 × 10 for human CD33, preferably the C2 domain of CD33 -7 M and below, Preferably 1 x 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 More than M Bottom, 5×10 -10 M or less, or 1 x 10 -10 This refers to an antibody that binds with a KD of M or less.

[0280] The antibodies or antigen-binding fragments described herein may exist in various forms, including those listed in Tables 39 and 4 0.

[0281] Described herein are 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. The CD33-specific antibodies or antigen-binding fragments exemplified herein are human. However, the exemplified antibodies or antigen-binding fragments may be chimerized.

[0282] In some embodiments, the CDR1, CDR2, and CDR3 sequences of any one of the antibodies listed in Table 39 are and a heavy chain comprising CDR3. In some embodiments, the CDR1, CDR2, and CDR3 sequences of any one of the antibodies listed in Table 39 are and CDR3 of any one of the antibodies listed in Table 40. and a light chain comprising CDR3. can be.

[0283] In some embodiments, a CD33-specific antibody or antibody comprising a heavy chain variable region as set forth in Table 38. In some embodiments, antigen-binding fragments thereof are provided. CD33-specific antibodies or antigen-binding fragments thereof comprising the region are provided.

[0284] The heavy 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, CD33 bispecific antibodies In some embodiments, the effector arm is a CD3 arm. In some embodiments of the bispecific antibody, the CD3 arms each have the sequence of SEQ ID NO: 662 , 663, 664, 671, 673, and 670 HCDR1, HCDR2, and HCDR3 , LCDR1, LCDR2, and LCDR3. In some embodiments, the CD3 arm comprises the VH and VH sequences of SEQ ID NOs: 652 and 661, respectively. In some embodiments of the CD33xCD3 bispecific antibody, the CD3 archaeon comprises a VL and a VL. The system contains the HC and LC of SEQ ID NOs: 640 and 676, respectively.

[0285] In some embodiments of the CD33xCD3 bispecific antibody, the CD3 arms each HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, HCDR7, HCDR8, HCDR9, HCDR10, HCDR11, HCDR12, HCDR13, HCDR14, HCDR15, HCDR16, HCDR17, HCDR18, HCDR19, HCDR19, HCDR110, HCDR111, HC Contains R2, HCDR3, LCDR1, LCDR2, and LCDR3. CD33×CD3 In some embodiments of the bispecific antibody, the CD3 arms are SEQ ID NOs: 657 and 658, respectively. and 678 VH and VL. In the example, the CD3 arm comprises the HC and LC of SEQ ID NOs: 675 and 677, respectively.

[0286] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 267, 26 0, 275, 270, 262, 258, 257, 281, 292, 291, 261, 26 9, 280, 259, 263, 264, 265, 266, 272, 277, 279, 28 a heavy chain variable region having a polypeptide sequence at least 95% identical to 4, or 285; or SEQ ID NOs: 287, 314, 309, 301, 298, 297, 290, 332, 33 1, 302, 310, 320, 300, 304, 305, 306, 307, 317, 31 9, 324, or 325. and wherein the anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 257 and SEQ ID NO: 258. 8, or a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 29 8 or 299. nothing.

[0287] IL1RAP specific antibody As used herein, the term "interleukin-1 receptor accessory protein" refers to a protein that is a protein that binds to an interleukin-1 receptor. "," "IL1RAP," and "IL1-RAP" are specifically listed in, for example, GenBan k Accession No. AAB84059, NCBI Reference S sequence:NP_002173.1, and UniProtKB / Swiss-Pr and human IL-1 as described in Accession No. Q9NPH3-1. 1RAP protein (SEQ ID NO: 576) (Huang et al., 1997, Proc.Natl.Acad.Sci.USA.94(24),12829-1283 2). IL1RAP has also been identified in the scientific literature as IL1 R3, C3orf13, and F Also known as LJ37788, IL-1 RAcP and EG3556.

[0288] The antibodies or antigen-binding fragments described herein may exist in a variety of forms, including those listed in Table 29. Contains one or more of the antibody CDRs shown.

[0289] As used herein, a recombinant antibody and an antigen-binding flag that specifically bind to IL1RAP are provided. In some embodiments, an IL1RAP-specific antibody or antigen binding The fragment is human IgG or a derivative thereof. The antibodies or antigen-binding fragments are human, although exemplified antibodies or antigen-binding fragments are chimerized. It may be done.

[0290] In some embodiments, the CDR1, CDR2, and CDR3 sequences of any one of the antibodies listed in Table 29 are and a heavy chain comprising CDR3. In some embodiments, the CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR110, CDR111, CDR12, CDR13, CDR14, CDR15 a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 29; 2, and a light chain comprising CDR3, and an IL1RAP-specific antibody or antigen-binding fragment thereof. is provided.

[0291] In some embodiments, the antibody comprises a heavy chain variable region of any one of the antibodies shown in Table 30. In some embodiments, an IL1RAP-specific antibody or antigen-binding fragment thereof is provided. an IL1RAP-specific antibody comprising the light chain variable region of any one of the antibodies shown in Table 30; or an antigen-binding fragment thereof. In some embodiments, an antibody of Table 30 is provided. An IL1RAP-specific antibody or its derivatives, comprising any one of a heavy chain variable region and a light chain variable region. Antigen-binding fragments are provided.

[0292] The heavy and light chain variable domains of the antibodies discussed in this section and shown in Table 30 , suitable for inclusion in a bispecific construct in which the targeting arm is an anti-IL1RAP arm For example, in some embodiments of the IL1RAP bispecific antibody, the effector The arm is a CD3 arm. Some embodiments of the IL1RAP x CD3 bispecific antibody In this embodiment, the CD3 arm is represented by SEQ ID NOs: 662, 663, 664, 671, 673, and 670 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LC In some embodiments of the IL1RAP x CD3 bispecific antibody, the CD3 The arms contain the VH and VL sequences of SEQ ID NOs: 652 and 661, respectively. IL1RAP×C In some embodiments of the D3 bispecific antibody, the CD3 arms each have the sequence of SEQ ID NO: 64 Contains 0 and 676 HC and LC.

[0293] In some embodiments of the IL1RAPxCD3 bispecific antibody, the CD3 arm is HCDR1, HDR2, HDR3, HDR4, HDR5, HDR6, HDR7, HDR8, HDR9, HDR10, HDR11, HDR12, HDR13, HDR14, HDR15, HDR16, HDR17, HDR18, HDR19, HDR19, HDR11, HDR12, HDR13, HDR14, HDR15, HDR16, HDR17, Contains CDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments of the xCD3 bispecific antibody, the CD3 arms are each represented by SEQ ID NO: The VH and VL of IL1RAP×CD3 bispecific antibodies are In some embodiments, the CD3 arm comprises the HC and LC of SEQ ID NOs: 675 and 677, respectively. Includes.

[0294] TMEFF2 specific antibody The present invention provides an isolated anti-TMEFF antibody that binds to the membrane proximal region of TMEFF2, SEQ ID NO: 629. The present invention provides an antibody or antigen-binding fragment thereof that binds to the membrane proximal region of TMEFF2. Anti-TMEFF2 antibodies are not internalized by cells. Without being bound to any particular theory, Although not intended to be a substitute for internalizing anti-TMEFF2 antibodies, non-internalizing anti-TMEFF2 antibodies are Antibody effectors resulting from the lack of internalization and degradation of TMEFF2 compared to antibodies It can be expected that the oncogenic effects mediated by the -function are improved.

[0295] "Binds to the membrane-proximal region" was identified using hydrogen / deuterium exchange (H / D exchange). This means that 90% of the antibody epitope residues are located within the membrane-proximal region of TMEFF2. Epitope residues are separated by at least a 5% difference in deuteration level due to H / D exchange. The test antibody is one that is protected. Exemplary such antibodies are the T MEB675, TMEB570, TMEB674, TMEB565, TMEB762, and and TMEB757.

[0296] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises an antibody having a sequence similar to residues HG KCEHSINMQEPSC (SEQ ID NO: 592) or DAGYTGQHCEKKDYSV L (SEQ ID NO: 600) binds to the membrane proximal region of TMEFF2. Residues HGKCEHSI An exemplary anti-TMEFF2 antibody that binds within NMQEPSC (SEQ ID NO: 592) is B570. Binding within residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 600) An exemplary anti-TMEFF2 antibody is TMEB675. EB762 and TMEB757 also contain residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 1). 600) is predicted to bind to the membrane-proximal region of TMEFF2.

[0297] In the H / D exchange assay, recombinantly expressed TMEFF2 ECD was lysed in the presence of antibodies. Incubation in deuterated water for a predetermined time in the presence or absence of Deuterium is incorporated at exchangeable hydrogen atoms that are not protected by the protein. Proteins are digested with proteases and the peptide fragments are analyzed using LC-MS. Recombination assays can be performed using known protocols. An exemplary protocol is shown below. This is described in Example 5.

[0298] The present invention also provides a heavy chain variable region (VH) of SEQ ID NO: 25 and a light chain variable region (VH) of SEQ ID NO: 28. VL of SEQ ID NO: 589 and VL of SEQ ID NO: 29, VH of SEQ ID NO: 27 and VL of SEQ ID NO: 29 VL of sequence number 30, VH of sequence number 589, VL of sequence number 31, VH of sequence number 604 and VL of SEQ ID NO: 607, or VH of SEQ ID NO: 612 and VL of SEQ ID NO: 613. An isolated anti-TMEFF antibody that competes with a reference antibody containing TMEFF2 for binding to the membrane-proximal region of TMEFF2. 2 antibodies or antigen-binding fragments thereof are provided.

[0299] Competition with a reference antibody for binding to the membrane-proximal region of TMEFF2 was measured using well-known methods. The method can be used to assay in vitro, for example, in the presence of unlabeled antibody. MSD Sulfo-tag™ NHS-ester labeled test antibody for TMEFF2 Binding to the membrane proximal region can be assessed by ELISA or by Biaco Competition can also be demonstrated using reassay or flow cytometry. the antibody inhibits binding of the reference antibody to the membrane proximal region of TMEFF2 by 85% or more, e.g., 90% or more; or inhibits by 95% or more, the test antibody competes with the reference antibody for binding to TMEFF2. is doing.

[0300] The present invention also relates to a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, HCDR7, HCDR8, HCDR9, HCDR10, HCDR11, HCDR12, HCDR13, HCDR14, HCDR15, HCDR16, HCDR17, HCDR18, HCDR19, HCDR110, 3. Isolated anti-T antibody containing light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. MEFF2 antibodies or antigen-binding fragments thereof are provided: SEQ ID NOs: 582, 584, 587, 18, 588, and 22, respectively; SEQ ID NOs: 583, 585, 16, 19, 21, and 23, respectively; SEQ ID NOs: 582, 586, 17, 18, 588, and 24, respectively; SEQ ID NOs: 583, 585, 16, 18, 588, and 22, respectively; or SEQ ID NOs: 582, 584, 587, 18, 588, and 603, respectively.

[0301] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof of the invention comprises: Approximately 0.4×10 -9 The equilibrium dissociation constant (K D ) binds to the membrane-proximal region of TMEFF2 , K. D Surface plasmon resonance was used in acetate buffer at pH 4.5-5.0 at room temperature. It is measured using

[0302] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof has a saturation of about 0.1 x10 -10 M ~ approx. 0.4×10 -9 K of M D It binds to the membrane-proximal region of TMEFF2.

[0303] The affinity of the antibody for the membrane-proximal region of TMEFF2 can be experimentally determined using any suitable method. Exemplary methods include ProteOn XPR36, BioCore re3000 or KinExA instrument, ELISA, or competitive binding assays known to those skilled in the art. The affinity of an antibody to TMEFF2 can be measured under different conditions (e.g., Therefore, affinity and other binding properties may differ when measured under different conditions (e.g., osmolality, pH). The integration parameter (e.g., K D , K. on , Koff) measurements are typically performed using standardized The conditions and buffers used are standardized, such as those described herein. Internal error in affinity measurements using Biacore 3000 or ProteOn ( The standard deviation (SD) is typically measured within the typical detection range. It will be understood by those skilled in the art that the range of "about 5 to 33%" may be used. The term "K D When values ​​are mentioned, they reflect the typical standard deviation in the assay. For example, K D is 1×10 -9 Typical SD for M is up to ±0.33×10 -9 M is.

[0304] The present invention also relates to VH3_3-23 (SEQ ID NO: 53) or VH1_1-69 (SEQ ID NO: The membrane-proximal region of TMEFF2, including the heavy chain variable region (VH) framework derived from The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to this region.

[0305] The present invention also relates to VKI_L11 (SEQ ID NO: 55) or VKIIII_A27 (SEQ ID NO: The membrane-proximal region of TMEFF2 contains the light chain variable region (VL) framework derived from the IL-1111 gene (No. 591). The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to a TMEFF2 site region.

[0306] The present invention also provides VH3_3-23 of SEQ ID NO: 53 or VKI of SEQ ID NO: 55, respectively. TMEFF2, including VH and VL frameworks derived from L11 An isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to the membrane proximal region is provided.

[0307] The present invention also relates to VH1_1-69 of SEQ ID NO: 54 and VK1_1-69 of SEQ ID NO: 591, respectively. TMEF, containing the VH and VL frameworks derived from III_A27 An isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to the membrane proximal region of F2 is provided.

[0308] The present invention also provides VH1_1-69 of SEQ ID NO: 54 and VKI of SEQ ID NO: 55, respectively. TMEFF2, including VH and VL frameworks derived from L11 An isolated anti-TMEFF2 antibody or antigen-binding fragment thereof that binds to the membrane proximal region is provided.

[0309] Antibodies containing heavy or light chain variable regions "derived from" particular framework or germline sequences The organism may be derived from a system that uses human germline immunoglobulin genes, e.g., the immunoglobulins discussed herein. Transgenic mice, rats, or chickens, or phage display lines It refers to antibodies obtained from a specific framework derived from germline sequences. Antibodies containing The sequence may contain amino acid differences when compared to the resulting sequence.

[0310] The present invention also relates to SEQ ID NOs: 582, 584, 587, 18, 588, and 22, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of The present invention provides an anti-TMEFF2 antibody or an antigen-binding fragment thereof.

[0311] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 25 VH and VL of SEQ ID NO:28.

[0312] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO: 39. and the VL is encoded by the polynucleotide of SEQ ID NO:42.

[0313] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: It contains HC of SEQ ID NO: 32 and LC of SEQ ID NO: 35.

[0314] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:46. and the VL is encoded by the polynucleotide of SEQ ID NO:49.

[0315] The present invention also provides the H of SEQ ID NOs: 583, 585, 16, 19, 21, and 23, respectively. including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, An isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is provided.

[0316] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 589 VH and SEQ ID NO:29 VL.

[0317] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO: 40. and the VL is encoded by the polynucleotide of SEQ ID NO:43.

[0318] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: It contains HC of SEQ ID NO: 33 and LC of SEQ ID NO: 36.

[0319] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:47. The LC is encoded by the polynucleotide of SEQ ID NO:50.

[0320] The present invention also relates to the antibodies of SEQ ID NOs: 582, 586, 17, 18, 588, and 24, respectively. Contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof.

[0321] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 27 VH and VL of SEQ ID NO: 30.

[0322] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO: 41. and the VL is encoded by the polynucleotide of SEQ ID NO:44.

[0323] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: It contains an HC of SEQ ID NO: 34 and an LC of SEQ ID NO: 37.

[0324] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:48. The LC is encoded by the polynucleotide of SEQ ID NO:51.

[0325] The present invention also relates to the antibodies of SEQ ID NOs: 583, 585, 16, 18, 588, and 22, respectively. Contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof.

[0326] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 589 VH and SEQ ID NO: 31 VL.

[0327] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO: 40. and the VL is encoded by the polynucleotide of SEQ ID NO:45.

[0328] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: It contains HC of SEQ ID NO: 33 and LC of SEQ ID NO: 38.

[0329] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:47. The LC is encoded by the polynucleotide of SEQ ID NO:590.

[0330] The present invention also relates to SEQ ID NOs: 582, 584, 587, 18, 588, and 60, respectively. 3 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising:

[0331] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 604 and VL of SEQ ID NO: 607.

[0332] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO: 618. and the VL is encoded by the polynucleotide of SEQ ID NO:619.

[0333] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 614 HC and SEQ ID NO: 615 LC.

[0334] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO: 620. The LC is encoded by the polynucleotide of SEQ ID NO:621.

[0335] The present invention also relates to SEQ ID NOs: 582, 584, 587, 18, 588, and 60, respectively. 3 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising:

[0336] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 612 and VL of SEQ ID NO: 613.

[0337] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO: 622. and the VL is encoded by the polynucleotide of SEQ ID NO:623.

[0338] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 616 HC and SEQ ID NO: 617 LC.

[0339] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO: 624. The LC is encoded by the polynucleotide of SEQ ID NO:625.

[0340] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is a multispecific antibody. It is a sexual antibody.

[0341] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is a bispecific antibody. It is a sexual antibody.

[0342] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof , which binds to T cell antigens.

[0343] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof , binds to CD3.

[0344] In some embodiments, an isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds to CD3ε.

[0345] VH, VL, HCDR, LCDR, HC, and The LC sequences are shown in Tables 60-67.

[0346] The embodiment described in the Examples has two variable domains, one derived from the heavy chain and one derived from the light chain. Although the present invention includes a pair of heavy or light chain variable domains, those skilled in the art will recognize that alternative embodiments include a single heavy or light chain variable domain. It will be appreciated that a single variable domain may be used to bind to TMEFF2. a variable domain capable of forming a two-domain specific antigen-binding fragment capable of Screening can be carried out, for example, by screening for the following compounds: The hierarchical dual combinatorial approach disclosed in JP 2002 / 01047 A phage display screening method using a dual combinatorial approach was used to In this approach, either the VH or VL chain clones can be used. Individual colonies containing the VL or VH chain were used to identify the complete clone encoding the other chain (VL or VH). The resulting two-chain specific antigen-binding domains were isolated by known methods and the present invention. Selection is made according to phage display techniques using those described in the specification. The individual VH and VL polypeptide chains are as disclosed in WO 1992 / 01047. The methods described herein are useful for identifying additional anti-TMEFF2 antibodies.

[0347] Bispecific anti-TMEFF2 / anti-CD3 antibody The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the an isolated bispecific antibody or an antibody thereof, wherein the antibody binds to the membrane proximal region of TMEFF2; Without being bound by any particular theory, TMEFF Bispecific antibodies that bind to the membrane-proximal region of 2 play a key role in mediating T cell-mediated killing of tumor cells. This can be more efficient.

[0348] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. An isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprising a Therefore, the antibody comprises a heavy chain variable region (VH) of SEQ ID NO: 25 and a light chain variable region (VH) of SEQ ID NO: 28. VL), VH of SEQ ID NO: 589 and VL of SEQ ID NO: 29, VH of SEQ ID NO: 27 and VL of SEQ ID NO: VL of SEQ ID NO: 30, VH of SEQ ID NO: 589, VL of SEQ ID NO: 31, VH of SEQ ID NO: 604 and and VL of SEQ ID NO: 607, or VH of SEQ ID NO: 612 and VL of SEQ ID NO: 613. an isolated bispecific antibody or antibody that competes with a control antibody for binding to the membrane-proximal region of TMEFF2; provides antigen-binding fragments thereof.

[0349] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen The binding fragment is approximately 0.4 × 10 -9 The dissociation constant (K D ) in the membrane-proximal region of TMEFF2 binds to K D is a surface plasmon resonance (SPRF) signal in an acetate buffer solution at pH 4.5-5.0 at room temperature. It is measured using resonance.

[0350] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen The binding fragment is approximately 0.1 × 10 -10 M ~ approx. 0.4×10 -9 K of M DTMEFF2 membrane Binds to the proximal region.

[0351] The present invention also relates to a method for producing a polypeptide comprising the following polypeptides: Isolated bispecific anti-TMEFF2 / anti-LCDR1 comprising CDR1, LCDR2, and LCDR3 3. Provide an antibody or antigen-binding fragment thereof: SEQ ID NOs: 582, 584, 587, 18, 588, and 22, respectively; SEQ ID NOs: 583, 585, 16, 19, 21, and 23, respectively; SEQ ID NOs: 582, 586, 17, 18, 588, and 24, respectively. SEQ ID NOs: 583, 585, 16, 18, 588, and 22, respectively; or SEQ ID NOs: 582, 584, 587, 18, 588, and 603, respectively.

[0352] The present invention also relates to a method for producing a medicament comprising administering to a subject the steps of: HCDR1, HCDR2, HCDR3, LCDR1, LCDR1 of 1, 673, and 690 2, and LCDR3, or an antigen thereof Provide a binding fragment.

[0353] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-TMEFF2 antibody, wherein the first domain comprises: Provided are: a CD3 antibody or antigen-binding fragment thereof; VH of SEQ ID NO: 25 and VL of SEQ ID NO: 28, VH of SEQ ID NO: 589 and VL of SEQ ID NO: 29; VH of SEQ ID NO: 27 and VL of SEQ ID NO: 30; VH of SEQ ID NO: 589 and VL of SEQ ID NO: 31; VH of SEQ ID NO: 604 and VL of SEQ ID NO: 607, or VH of sequence number 612 and VL of sequence number 613.

[0354] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-TMEFF2 antibody, wherein the second domain comprises: Provided are: a CD3 antibody or antigen-binding fragment thereof; VH of sequence number 652 and VL of sequence number 661.

[0355] In some embodiments, the second domain comprises a VH of SEQ ID NO: 657 and a VH of SEQ ID NO: 65 Includes 8 VLs.

[0356] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the There was, The first domain is set forth in SEQ ID NOs: 582, 584, 587, 18, 588, and and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR22 3, wherein the second domain comprises SEQ ID NOs: 662, 663, 664, 671, 673 and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first domain comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28, and the second domain comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28. comprising a VH of SEQ ID NO: 652 and a VL of SEQ ID NO: 661; The first domain comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28, and the second domain comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28. comprising a VH of SEQ ID NO: 657 and a VL of SEQ ID NO: 678; The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 32 HC1 of SEQ ID NO: 35, LC1 of SEQ ID NO: 640, and LC of SEQ ID NO: 676 Contains 2; and / or The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 32 HC1 of SEQ ID NO: 35, LC1 of SEQ ID NO: 675, and LC of SEQ ID NO: 677 2.

[0357] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the There was, The first domains are set forth in SEQ ID NOs: 583, 585, 16, 19, 21, and 2, respectively. 3 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 wherein the second domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 67 3, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and Contains LCDR3; the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 29, 2 domains comprising a VH of SEQ ID NO: 652 and a VL of SEQ ID NO: 661; the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 29, the two domains comprising a VH of SEQ ID NO: 657 and a VL of SEQ ID NO: 678; and / or The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 33 HC1 of SEQ ID NO: 36, LC1 of SEQ ID NO: 640, and LC of SEQ ID NO: 676 Includes 2; The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 33 HC1 of SEQ ID NO: 36, LC1 of SEQ ID NO: 675, and LC of SEQ ID NO: 677 2.

[0358] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the There was, The first domain is set forth in SEQ ID NOs: 582, 586, 17, 18, 588, and 24 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 and the second domain comprises SEQ ID NOs: 662, 663, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 73, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3; The first domain comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30, and the second domain comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30. comprising a VH of SEQ ID NO: 652 and a VL of SEQ ID NO: 661; The first domain comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30, and the second domain comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30. comprising a VH of SEQ ID NO: 657 and a VL of SEQ ID NO: 678; and / or The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 34 HC1 of SEQ ID NO: 37, LC1 of SEQ ID NO: 640, and LC of SEQ ID NO: 676 Includes 2; The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 34 HC1 of SEQ ID NO: 37, LC1 of SEQ ID NO: 675, and LC of SEQ ID NO: 677 2.

[0359] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the There was, The first domain is set forth in SEQ ID NOs: 583, 585, 16, 18, 588, and 22 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 and the second domain comprises SEQ ID NOs: 662, 663, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 73, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3; the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 31, 2 domains comprising a VH of SEQ ID NO: 652 and a VL of SEQ ID NO: 661; the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 31, the two domains comprising a VH of SEQ ID NO: 657 and a VL of SEQ ID NO: 678; and / or The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 33 HC1 of SEQ ID NO: 38, LC1 of SEQ ID NO: 640, and LC of SEQ ID NO: 676 Includes 2; The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 33 HC1 of SEQ ID NO: 38, LC1 of SEQ ID NO: 675, and LC of SEQ ID NO: 677 2.

[0360] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the There was, The first domain is set forth in SEQ ID NOs: 582, 584, 587, 18, 588, and and 603 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCD R3, wherein the second domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, and 671, respectively. , 673, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and LCDR3; the first domain comprises a VH of SEQ ID NO: 604 and a VL of SEQ ID NO: 607, the second domain comprises a VH of SEQ ID NO: 652 and a VL of SEQ ID NO: 661; the first domain comprises a VH of SEQ ID NO: 604 and a VL of SEQ ID NO: 607, the second domain comprises a VH of SEQ ID NO: 657 and a VL of SEQ ID NO: 678; and / or teeth The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 61 4, LC1 of SEQ ID NO: 615, HC2 of SEQ ID NO: 640, and Contains LC2; The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 61 4, LC1 of SEQ ID NO: 615, HC2 of SEQ ID NO: 675, and An isolated bispecific antibody or antigen-binding fragment thereof comprising LC2 is provided.

[0361] The present invention also provides a method for detecting a leukocyte-associated leukemia virus (LEV) comprising administering to a subject a first domain that binds to TMEFF2 and a second domain that binds to CD3. an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, comprising the There was, The first domain is set forth in SEQ ID NOs: 582, 584, 587, 18, 588, and and 603 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCD R3, wherein the second domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, and 671, respectively. , 673, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 , and LCDR3; the first domain comprises a VH of SEQ ID NO: 612 and a VL of SEQ ID NO: 613, the second domain comprises a VH of SEQ ID NO: 652 and a VL of SEQ ID NO: 661; the first domain comprises a VH of SEQ ID NO: 612 and a VL of SEQ ID NO: 613, the second domain comprises a VH of SEQ ID NO: 657 and a VL of SEQ ID NO: 678; and / or teeth The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 61 6 HC1, SEQ ID NO: 617 LC1, SEQ ID NO: 640 HC2, and SEQ ID NO: 676 Contains LC2; The bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 61 6 HC1, SEQ ID NO: 617 LC1, SEQ ID NO: 675 HC2, and SEQ ID NO: 677 An isolated bispecific antibody or antigen-binding fragment thereof comprising LC2 is provided.

[0362] Embodiments: The present invention provides the following non-limiting embodiments. 1. An isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof, comprising: a) heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 662, heavy chain complementarity determining region (HCDR) 2 comprising SEQ ID NO: 663 a heavy chain comprising an HCDR2 comprising SEQ ID NO: 664, and an HCDR3 comprising SEQ ID NO: 671 a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 673, a light chain complementarity determining region (LCDR) 2 comprising SEQ ID NO: 674, and a light chain complementarity determining region (LCDR) 3 comprising SEQ ID NO: 675, a light chain complementarity determining region (LCDR) 4 comprising SEQ ID NO: 676, a light chain complementarity determining region (LCDR) 5 comprising SEQ ID NO: 677, a light chain complementarity determining a light chain containing LCDR3 containing number 690; b) a heavy chain variable region comprising SEQ ID NO: 652 and a light chain variable region comprising SEQ ID NO: 661; c) a heavy chain comprising SEQ ID NO: 640 and a light chain comprising SEQ ID NO: 676; d) HCDR1 comprising SEQ ID NO: 662, HCDR2 comprising SEQ ID NO: 663, and the sequence a heavy chain comprising an HCDR3 comprising SEQ ID NO: 664, and an LCDR1 comprising SEQ ID NO: 773, a light chain comprising LCDR2 comprising sequence number 673 and LCDR3 comprising sequence number 690; e) a heavy chain variable region comprising SEQ ID NO: 657 and a light chain variable region comprising SEQ ID NO: 678; Ruiha f) an isolated recombinant antibody comprising a heavy chain comprising SEQ ID NO: 675 and a light chain comprising SEQ ID NO: 678; CD3 antibody or its antigen-binding fragment. 2. A binding affinity of about 300 nM or less in cynomolgus monkeys (Macaca fascicularis) or humans Isolated recombinant antibody that specifically binds to CD3d, or CD3e, or both CD3e and CD3d An anti-CD3 antibody or an antigen-binding fragment thereof. 3. The isolated recombinant anti-CD20 antibody of embodiment 2, wherein the binding affinity is about 100 nM or less. 3An antibody or antigen-binding fragment thereof. 4. The binding affinity is determined by flow cytometry or Protein analysis at +25°C. Surface plasmon resonance assay ProteOn XPR36 system 4. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof of embodiment 2 or 3. 5. Any of embodiments 1-4, having one, two, three, or four of the following characteristics: 1. An isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof according to any one of the preceding claims. a) To human and cynomolgus monkey CD3+ T lymphocytes with a calculated EC50 of 300 nM or less Binds to cynomolgus CD3-expressing HEK cells with a calculated EC50 of 300 nM or less Calculation between binding to CD3+ T lymphocytes and binding to cynomolgus monkey CD3-expressing HEK cells If the difference in EC50 is less than 5-fold, calculate the EC50 using flow cytometry. as measured in whole cell binding assays; b) an equilibrium dissociation constant (K) of 300 nM or less D ) and human-derived recombinant CD3d (SEQ ID NO: 691), or recombinant human CD3e (SEQ ID NO: 636), or recombinant cytoplasmic CD3e (SEQ ID NO: 636). Binds to recombinant CD3d (SEQ ID NO: 692) from cynomolgus monkeys or recombinant CD3d from cynomolgus monkeys Binds to CD3e (SEQ ID NO: 693) and K D Proteon surface plasmon Measured using the ProteOn XPR36 system; c) binds to residues 1-6 of CD3e as determined by X-ray crystallography; or d) cOKT3 or SP34- as determined by fluorescence-activated cell sorting assay Activates T cells or induces CD69 expression to a similar extent as 2. 6. Any one of embodiments 1-5, comprising at least one substitution in an antibody constant domain. 10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the at least one substitution is a) comprising the heavy chain substitutions K409R, F405L, or F405L and R409K; b) comprising the heavy chain substitutions S228P, F234A, and L235A; c) heavy chain substitutions L234A, G237A, P238S, H268A, A330S, and P331S, wherein the antibody is of the IgG1 isotype; or d) comprising the heavy chain substitution S228P, wherein the antibody is of the IgG4 isotype; An antibody or antigen-binding fragment thereof, wherein residue numbering is according to the EU index. 7. HCDs of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, The antibody or antigen-binding fragment thereof according to any one of aspects 1 to 6. 8. Heavy chain variable region (VH) and light chain variable region (VH) of SEQ ID NOs: 652 and 661, respectively. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, comprising: 9. A antibody containing the heavy chain sequence (HC) and light chain sequence (LC) of SEQ ID NOs: 640 and 676, respectively. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8. 10. HCs of SEQ ID NOs: 662, 663, 664, 773, 673, and 690, respectively HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5. 11. Any of embodiments 1 to 5, comprising a VH and a VL of SEQ ID NOs: 657 and 678, respectively. The antibody or antigen-binding fragment thereof described in any one of the above. 12. Any of embodiments 1 to 5, comprising the HC and LC of SEQ ID NOs: 675 and 677, respectively. The antibody or antigen-binding fragment thereof described in any one of the above. 13. HCs of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. or an antigen-binding fragment thereof. 14. An antibody or antigen-binding fragment thereof, comprising the VH and VL sequences of SEQ ID NOs: 652 and 661, respectively. Combined piece. 15. An antibody or its antigen-binding fragment, comprising the HC and LC of SEQ ID NOs: 640 and 676, respectively. Combined piece. 16. HCs of SEQ ID NOs: 662, 663, 664, 773, 673, and 690, respectively HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. or an antigen-binding fragment thereof. 17. An antibody or antigen-binding fragment thereof, comprising VH and VL sequences of SEQ ID NOs: 657 and 678, respectively. Combined piece. 18. An antibody or its antigen-binding fragment, comprising the HC and LC of SEQ ID NOs: 675 and 677, respectively. Combined piece. 19. Any one of embodiments 1 to 18, wherein the antibody is human or humanized. The antibody or antigen-binding fragment thereof described in (1). 20. The method of embodiment 19, wherein the antibody is of the IgG4 or IgG1 isotype. antibody. 21. The antibody contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the Fc region. 21. The antibody of embodiment 20. 22. The antibody of embodiment 18, a) light chain substitutions of D43G, L49M, L50I, S62N, Q85E; b) D43G, V48L, L49M, L50I, S62N, Q85E, H89Y light vehicles strand displacement, c) heavy chain substitutions of R10G, R13K, V73I, R70K, T83S, L96V; d) light chain substitutions D43G, V48L, L49M, L50I, S62N, Q85E, or One of H89Y, or e) heavy chain substitutions R10G, R13K, V73I, R79K, T83S, or L96V Either one, Including, Residue numbering for light chain substitutions is in accordance with SEQ ID NO: 661, and for heavy chain substitutions is in accordance with SEQ ID NO: 65 2, according to the antibody. 23. Any one of embodiments 1 to 22, wherein the antibody is bispecific or multispecific. The antibody described in (1). 24. A first domain that specifically binds to CD3 and a second domain that specifically binds to a second antigen. a bispecific antibody comprising two domains, each of said first domains being represented by SEQ ID NO: 662, 663, 664, 671, 673, and 690 HCDR1, HCDR2, HC A bispecific antibody comprising LCDR1, LCDR2, and LCDR3. 25. The first domain and the second domain are of the IgG4 isotype, and the The first or second domain is S228P, F234A, L235A, F405L, and R4 The other of the first and second domains contains a heavy chain substitution of S228P. , F234A, and L235A heavy chain substitutions, residue numbering is according to the EU index 25. The bispecific antibody of embodiment 24. 26. The first and / or second domain comprises F405L, or F405L and R40 Contains at least one substitution in the CH3 constant domain, including a 9K substitution, and residue numbering is 25. The bispecific antibody of embodiment 24, wherein the IgG is in accordance with the EU index. 27. One of the first or second domains comprises a heavy chain substitution of F405L, and the first or the other of the second domains contains a heavy chain substitution of K409R, and the residue numbering is in accordance with the EU index. 25. The bispecific antibody of embodiment 24, which is in accordance with 28. The first domain and the second domain are of the IgG4 isotype, and the One of the first or second domains contains a heavy chain substitution of S228P, and the first or second domain The other main one contains heavy chain substitutions of S228P, F405L, and R409K, with the addition of residues 25. The bispecific antibody of embodiment 24, wherein the numbering is according to the EU index. 29. The first domain comprises a VH and a VL of SEQ ID NOs: 652 and 661, respectively. 25. The bispecific antibody of claim 24. 30. The first domain comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively. 25. The bispecific antibody of claim 24. 31. The first domain comprises a VH and a VL of SEQ ID NOs: 657 and 678, respectively. 25. The bispecific antibody of claim 24. 32. The first domain comprises the HC and LC of SEQ ID NOs: 675 and 677, respectively. 25. The bispecific antibody of claim 24. 33. The second antigen is expressed on a cell surface of a target cell other than an immune effector cell. 25. The bispecific antibody of claim 24, which is an antigen. 34. The bispecific antibody of claim 33, wherein the cell surface antigen is a tumor-associated antigen. . 35. The second antigen is CD33, IL1RAP, PSMA, or TMEFF2. The bispecific antibody according to any one of claims 24 to 34, 36. The first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 6 73, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3, wherein the second domain is set forth in SEQ ID NOs: 78, 683, 80, and 8 HCDR1, HCDR2, HCDR3, LCDR1, LCDR for 1, 792, and 686 36. The bispecific antibody of embodiment 35, comprising LCDR1, LCDR2, and LCDR3. 37. The first domain comprises a VH and a VL of SEQ ID NOs: 652 and 661, respectively. wherein the second domain comprises a VH and a VL of SEQ ID NOs: 681 and 682, respectively; 34. The bispecific antibody of embodiment 33. 38. The first domain comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively. wherein the second domain comprises the HC and LC of SEQ ID NOs: 679 and 680, respectively; 34. The bispecific antibody of embodiment 33. 39. The first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 6 73, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3, wherein the second domain is set forth in SEQ ID NOs: 78, 3, 80, 81, 792 and 686 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. 40. The first domain comprises the VH and VL of SEQ ID NOs: 652 and 661, respectively. and the second domain comprises the VH and VL of SEQ ID NOs: 8 and 682, respectively. 34. The bispecific antibody of claim 33. (margin). 41. The first domain comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively. and the second domain comprises the HC and LC of SEQ ID NOs: 13 and 680, respectively. 34. The bispecific antibody of embodiment 33. 42. The first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 6 73, and 690 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3, wherein the second domain is set forth in SEQ ID NOs: 78, 79, 80, and 81, respectively. , 82, and 83 of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3. 43. The first domain comprises a VH and a VL of SEQ ID NOs: 652 and 661, respectively. wherein the second domain comprises a VH and a VL of SEQ ID NOs: 116 and 117, respectively; 34. The bispecific antibody of embodiment 33. 44. The first domain comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively. wherein the second domain comprises the HC and LC of SEQ ID NOs: 138 and 139, respectively; 34. The bispecific antibody of embodiment 33. 45. The first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 6 73, and 670 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3, wherein the second domain is set forth in SEQ ID NOs: 164, 165, and 166, respectively. , 167, 168, and 169 of HCDR1, HCDR2, HCDR3, LCDR1, L 32. The bispecific antibody of embodiment 31, comprising CDR2, and LCDR3. 46. ​​The first domain comprises a VH and a VL of SEQ ID NOs: 652 and 661, respectively. wherein the second domain comprises a VH and a VL of SEQ ID NOs: 215 and 216, respectively; 32. The bispecific antibody of embodiment 31. 47. The first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 6 73, and 670 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3, wherein the second domain is set forth in SEQ ID NOs: 349, 390, and 341, respectively. , 471, 513, and 555 HCDR1, HCDR2, HCDR3, LCDR1, L 32. The bispecific antibody of embodiment 31, comprising CDR2, and LCDR3. 48. The first domain comprises a VH and a VL of SEQ ID NOs: 652 and 661, respectively. wherein the second domain comprises a VH and a VL of SEQ ID NOs: 267 and 306, respectively; 32. The bispecific antibody of embodiment 31. 49. The first domain is selected from the group consisting of SEQ ID NOs: 662, 663, 664, 671, and 6 73, and 670 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and and LCDR3, wherein the second domain is selected from the group consisting of SEQ ID NOs: 363, 404, and 445, respectively. , 485, 527, and 569 HCDR1, HCDR2, HCDR3, LCDR1, L 32. The bispecific antibody of embodiment 31, comprising CDR2, and LCDR3. 50. The first domain comprises the VH and VL of SEQ ID NOs: 652 and 661, respectively. wherein the second domain comprises a VH and a VL of SEQ ID NOs: 281 and 320, respectively; 32. The bispecific antibody of embodiment 31. 51. An antibody according to any one of embodiments 1 to 50 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: 52. A polynucleoside encoding an antibody according to any one of embodiments 1 to 50. Chid. 53. A vector comprising the polynucleotide of embodiment 52. 54. A host cell comprising the vector of embodiment 53. 55. A method for producing an antibody according to any one of embodiments 1 to 50, comprising: Culturing the host cell of embodiment 52 under conditions in which the polypeptide is expressed by the host cell; and recovering the antibody produced thereby. 56. A method of treating cancer in a subject, comprising administering a compound to a subject for a time sufficient to treat the cancer. and administering to said subject in need thereof a therapeutically effective amount of any one of embodiments 1 to 50. 2. A method comprising administering an isolated antibody according to claim 1. 57. The method of embodiment 56, wherein the cancer is a solid tumor or a hematological malignancy. 58. The solid tumor is prostate cancer, colorectal cancer, gastric cancer, renal clear cell carcinoma, bladder cancer, lung cancer, or tonsillitis. Squamous cell carcinoma, glioma, breast cancer, kidney cancer, angiogenesis disorders, clear cell renal carcinoma (CCRCC), pancreatic cancer 58. The method of embodiment 57, wherein the cancer is renal carcinoma, urothelial carcinoma, or liver adenocarcinoma. 59. The prostate cancer is refractory prostate cancer, prostatic intraepithelial neoplasia, androgen-independent pre- 59. The method of embodiment 58, wherein the cancer is prostate cancer or malignant prostate cancer. 60. 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) , The method of embodiment 57. 61. Any of embodiments 56-60, wherein the antibody is administered in combination with a second therapeutic agent. The method according to any one of the preceding claims. 62. The antibody of any one of embodiments 1 to 50, for use in therapy. 63. An anti-idiotypic antibody that binds to an antibody according to any one of embodiments 1 to 50. . [Example]

[0363] 1. De novo generation and functional characterization of anti-CD3 mAb 1-1 OmniRat with CD3 antigen to generate CD3 monoclonal antibodies Immunization with s® OmniRats® was synthesized using human CD3e and human CD3d; cynomolgus monkey CD A proprietary vector (Aldevron, F) encoding 3e and cynomolgus monkey CD3d The animals were immunized with human and cynovirus-associated antibodies (Bergo, North Dakota, USA). From the sixth injection, animals were alternately boosted with rhesus monkey DNA. The optimized vector was administered. Lymph node derived cells were fused with the Ag8 myeloma cell line. Forty million cells from the fused BLW were cultured in three 96-well plates after IgM depletion. 133 million cells derived from the fusion BLX were placed on nine plates that were not depleted of IgM. Placed in a 96-well plate.

[0364] a) Screening vector: pOPT-CD3e-hum-epsilon-TCE .OMT+pOPT-CD3d-hum-delta.OMT, 1:1(pOPT-CD 3e / d-hum-mix), and pcDNA3.1-CD3e-cyn-delta+ pcDNA3.1-CD3d-cyn-delta, 1:1(pcDNA3.1-CD3 Human and cynomolgus monkey cDNA cloned into the cynomolgus monkey e / d-cyn-mix was transiently In transfected cells, fusion B was detected by cell-based ELISA (CELISA). LW (with magnetic bead depletion of lymphocytes) and BLX (without magnetic bead depletion of lymphocytes) The hybridoma supernatants from the human and cynomolgus monkey cDNA sequences (and corresponding The amino acid sequences are provided in Table 4. For the CELISA negative control, transfected Incubation of uncultured mammalian cells with hybridoma supernatant and secondary Bethy For the CELISA transfection control, the same conjugate as above was used. Mammalian cells transfected with the lactate were detected with an anti-tag antibody.

[0365] Hybridoma supernatants were cultured using CD3-positive and CD3-negative Jurkat cells: Jurkat CD3+ (E6-1) and Jurkat CD3- (J.RT3-T3.5) Further analysis was performed by flow cytometry (FACS). For the FACS negative control, D3-negative Jurkat cells (J.RT3-T3.5) were incubated with dilution buffer. Southern anti-rat IgHRP and Bethyl anti-rat IgG1, 2a, Detection was performed with 2b and 2c-HRP secondary antibodies.

[0366] Human and cynomolgus CD3e / d presented on transiently transfected cells For the complex, or in the case of human CD3e / d complex, Jurkat CD3+ (E6- 1) In cells, hybridomas derived from fusion BLW and fusion BLX The specificity of the antibodies in the supernatant was confirmed by CELISA on transiently transfected cells. This was demonstrated when tested by FACS on the Jurkat cell line (Table 3). A significant signal for any sample in the experimental samples served as a negative control. was not detected.

[0367] [Table 3]

[0368] [Table 4]

[0369] 1-2 Cloning anti-CD3 antibodies OmniRats (OMT, Palo Alto, California, USA) Anti-human CD3 antibodies were generated. The variable region ("V region") sequences of these clones were analyzed using genomic DNA. All sequences obtained were either human IgG heavy chain or λ light chain. The sequences, especially the LC, showed high homology. This revealed several mutations in the framework (Figure 1). and then synthesize the heavy chain sequence into a mammalian expression vector, the light chain sequence into a human IgG1 vector, and The seven mAbs were cloned into a λ vector. The sequences are shown in Tables 6 and 7. A separate code was assigned (Table 5A).

[0370] CD3B312 was selected as the most representative clone, followed by S228P, F234A, and L2 Human IgG1σ with the 35A mutation and assigned protein identifiers as shown in Table 5B The heavy chain sequences were cloned into IgG4PAA and IgG4PAA. These were used to construct bispecific antibodies. We generated T cells and demonstrated their T cell redirection function through cytotoxicity.

[0371] [Table 5]

[0372] [Table 6]

[0373] [Table 7-1]

[0374] [Table 7-2]

[0375] [Table 7-3]

[0376] [Table 8]

[0377] [Table 9]

[0378] 1-3 Screening of hybridomas for binding to purified human and cynomolgus monkey T cells Training Individual LA binding sites for purified human (Fig. 2) and cynomolgus monkey (Fig. 3) CD3+ T lymphocytes To evaluate the binding capacity of the hybridoma supernatants, a cell-based binding assay was designed. T cells were counted and diluted to 1×10^6 cells / mL and 0.5 μL / mL of Live / Dead Fixable Green Dead Cell Stain(Life The cells were then incubated with 100 μg of PBS (Technologies, L-2301). 100 μL / well (1×10^5 cells / well) in a U-bottom plate (Falcon 353077 The plate was centrifuged at 300 g for 5 minutes to pellet the cells. The supernatant was removed. The plate was vortexed briefly to resuspend the cells. The tumor supernatant was washed with FACS staining buffer (BSA, BD Biosciences 55465 7) to 4.5 μg / mL, and then dilute to a minimum concentration of 0.006 μg / mL. The positive control for mouse anti-human CD3 (SP34-2, B D Biosciences 551916) and a negative isotype control (mouse IgG 1 BD Biosciences 556648) was also diluted to 4.5 μg / mL. 100 μL of each sample was added to T cells and incubated at 4°C for 1 hour. Wash once with buffer and incubate with secondary AF647 goat anti-mouse IgG (Life Technology Antibodies, A21235) or AF647 goat anti-rat IgG (Life Technologies, ologies, A21247) with the appropriate species (anti-La for hybridoma samples) Antibodies were added at 10 μg / mL in 50 μL according to the protocol (anti-mouse for control antibodies). The plates were incubated at 4°C for 45 minutes and washed twice with staining buffer. 5 μL of running buffer (staining buffer + 1 mM EDTA (Life Technologies) ologies, AM9260G) + 0.1% Pluronic F-68 (Life Technologies Resuspend in chemistries 24040-032) and use Intellicyt system. The results are shown in Figures 2 and 3.

[0379] In other experiments, purified human T cells were plated at 1.1 × 10^5 cells / well in U-bottom plates. The plates were centrifuged at 300 g for 5 minutes to pellet the cells, and the supernatant The plate was briefly vortexed to resuspend the cells. Hybridoma The supernatant was stained with FACS staining buffer (BSA, BD Biosciences 554657). Dilute to 30 μg / mL with 1000 kcal / mL of HCl, then dilute to a minimum concentration of 0.00017 μg / mL. 11 serial dilutions were made at 1:3 dilutions. 50 μL of each sample was added to the T cells and incubated at 4°C for 1 hour. The cells were washed once with staining buffer and stained with secondary Dylight 65. 0 goat anti-rat IgG (Bethyl, A110-239D5) at 10 μg / mL in 50 μL The plate was incubated at 4°C for 1 hour and washed twice with staining buffer. Cells were resuspended in 30 μL of FACS buffer and analyzed using a Hypercyte flow cytometer. Binding to primary human T cells was measured using a cytochrome P450 meter (Intellicyt Corp.). Representative dose-response curves for anti-CD3 clones are shown in FIG.

[0380] SP34-2 (a commercially available antibody with a known epitope and cross-reactivity with cynomolgus monkey CD3) A representative competitive binding curve with the anti-human CD3 antibody (Figure 6) is shown. Initial screening results The results are summarized in Table 8. Six of the clones showed positive binding and also showed binding to primary human T cells. Competes with SP34-2 for binding.

[0381] 1-4 SP34-2 competitive assay with commercially available CD3 antibody In addition, a commercially available anti-human IgG antibody with a known epitope and cross-reactivity with cynomolgus monkey CD3 was also developed. Hybridoma supernatants were evaluated for their ability to compete with the human CD3 antibody SP34-2. First, a concentration titration curve of AF488 fluorescently labeled SP34-2 (BD, 557705) was performed. This determined the fixed concentration of SP34-2 for the following competition assay. Human purified T cells were diluted with PBS to 1×10^6 cells / mL. an TruStain Fc Block, Biolegend, 422302) 5 Add 100 μL / 100 μL cells and plate 100 μL / well in a U-bottom plate. AF488 SP34-2 and AF488-labeled isotype control (AF488 Mouse IgG1, BD, 400129) was diluted from 50 μg / mL to 0 μg / mL in a 1:2 dilution scheme. The plates were centrifuged at 300 x g for 5 minutes and the cells were serially diluted to 0.049 µg / mL. Pellet and remove supernatant. Gently vortex the plate to resuspend the cells. 50 μL of each AF488 SP34-2 dilution was added to the cells and incubated at 4°C for 1 hour. The plate was washed twice with staining buffer and twice with running buffer (staining buffer + 1mM EDTA(Life technologies, AM9260G)+0.1% Pluronic F-68 (Life Technologies 24040-032) The cells were resuspended in 25 μL of running buffer and run on an HTFC screen. The assay was read using a scanning system (IntelliCyt Corporation). For the competition assay, a fixed concentration of SP34-2 of 2 μg / mL was used based on the dose-response curve. was selected.

[0382] Seven hybridomas that demonstrated binding to purified T cells were compared with SPs that bind to human T cells. 34-2 (Figure 4). A control antibody was included in the competition assay. Labeled mouse anti-human CD3, SP34-2 antibody and mouse anti-human CD3, UCHT1 antibody As positive controls, rat IgG and mouse isotypes were used. Purified human T cells were used as a negative control for 4-2. 1×10^6 cells / mL in PBS. 5 μL of Fc block (Human TruS Contain Fc Block (Biolegend, 422302) and per 1 mL of cells 0.5 μL of Live / Dead Fixable Far Red Dead Cell Add ll Stain (Life Technologies L10120) to the cells The plate was then incubated at 4°C for 15 minutes. 5 cells (1×1 0^5 cells / well) into a 96-well U-bottom plate (Falcon 353077) The plate was centrifuged at 300 x g for 5 minutes to pellet the cells, and the supernatant was removed. The plate was gently vortexed to resuspend the cells. Hybridoma supernatant and Control antibodies were stained with FACS staining buffer (BSA, BD Biosciences 5546 57) to twice the desired final concentration. 35 μL of 2× hybridoma supernatant and control Mix the antibody with 35 μL of 2× AF488 SP34-2 (4 μg / mL) to obtain the desired 1 × concentration of hybridoma supernatant, 1× concentration of control antibody, and 2 μg / mL of AF488 S Hybridoma supernatants and control antibodies were titrated at a range of concentrations using a seven-point titration. The hybridoma supernatants were assayed at concentrations ranging from 200 μg / mL to 0.08 μg / mL. The control antibody was assayed from 100 μg / mL to 0.04 μg / mL. 1x hybridoma supernatant containing AF488 SP34-2 or 1x control antibody 5 μg / mL 0 μL was added to the T cells and incubated at 4°C for 2 hours. Twice, running buffer (staining buffer + 1 mM EDTA (Life Technologies) Logies, AM9260G) + 0.1% Pluronic F-68 (Life Tech The cells were washed once with 25 μL of Ranini. The cells were resuspended in the HPLC buffer and used in the HTFC screening system (IntelliCyt SP34-2 (having a known epitope, Representative competitive binding curves with a commercially available anti-human CD3 antibody that is cross-reactive with macaque CD3 The lines are shown in Figures 4 and 6. The initial screening results are summarized in Table 8. Six of the clones One showed positive binding and also competed with SP34-2 for binding to primary human T cells.

[0383] As shown in Figure 4, the seven antibodies competed with SP34-2 with similar curves. A rightward shift in the curve for 4-2 indicates weaker binding affinity. As expected, the antibody IgG did not compete with SP34-2.

[0384] [Table 10]

[0385] 1-5 T cell activation as measured by CD69 upregulation Screening of all hybridoma hits Hybridoma hits using primary human and cynomolgus monkey T cell-based assays The ability of these antibodies to activate T cells was determined, which mimics the cross-linking effect of TCR activation. This was achieved by coating the plate with antibodies. Cells are known to upregulate the surface expression of the protein, CD69. Unknown samples or controls (positive control: in-house, Okt-3 BISB264.002 , BD Bioscience SP-34-2 #551916; negative control: anti-CD2 50 μL of 10 μg / mL antibody preparation containing 0.0, in-house, BISB266.004) The experiment was carried out by coating 96-well plates (Costar #3361) with The experiment was carried out. The plates were incubated overnight at 4°C. The next day, the plates were washed with PBS for 2 min. Frozen primary T cells (human T cells were collected from Biological Specialty Cynomolgus monkeys were sourced from WorldWide Prim The cells were thawed, counted for viability, and cultured in RPMI 1640 medium ( in 10% HI FBS (Gibco #11875) containing Gibco #10062 to 2×10 6 100 μL of cells were added to the plate and incubated at 37°C. The plates were incubated overnight (approximately 16 hours) at 5% CO2. The cells were pelleted by spinning at 400 rpm for 3 min and the supernatant was discarded. The cells were washed once with PBS. , and rotated as previously described. Live / Dead Green Fixable in PBS 10 ml of a 2.5% solution of e Dye (Life Technologies #L23101) 1 μL was added to each well and incubated at room temperature in the dark for 10 minutes. Anti-CD69 AF488 ( Add 50 μL of a 1% solution of Biolegend #310916 Lot #B125271 The plate was incubated at 4°C for 45 minutes. The cells were pelleted as described above. Discard the supernatant and resuspend the cells in 150 μL of FACS buffer. After the final wash, the cells were resuspended in 150 μL of FACS buffer and The cells were read on an ACS Canto. As can be seen in Figure 20, the measured anti-CD69 staining The positive controls cOkt3 and SP34-2 were human SP34-2 induced upregulation of CD69 in T cells. Induced CD69 expression in macaque T cells, which is conserved from monkeys to humans The OKT3 anti-CD3 clone binds to a region of the CD3 sequence. D3 and did not induce upregulation of CD69. The negative control for both human and monkey T cells was anti-CD20, which is not expressed on T cells. Several of the hybridoma clones tested for cell activation showed activity similar to that of the positive control. To the extent of, i.e., 2B4, 2E6, 3B4, 1F8, 2E9, 3F4, 3G8, 4E 5, 5H7, 8B4, 8G8, and 1F1 induced CD69 expression to a similar extent. Most of them, except for 5H7, 8B4, and 8G8, bound to cynomolgus monkey T cells. It was activated.

[0386] 1-6 Framework genetic manipulation of BLW-2E6 The clones show high homology compared to human immunoglobulin germline sequences and Clone 2E6 was selected and subjected to standard PCR amplification. The framework sequences were matched. Six mutations in HC and seven mutations in LC were All mutations were backmutated individually or in combination to the human germline sequence (Table 9). V-region DNA sequences were synthesized and cloned into the same mammalian expression vector as the parent construct. The HC and LC constructs were paired in a matrix format to Proteins with individual or combinations of mutations were generated and protein activity was tested. V48 in LC could not be reverted to the germline. All other revertants The mutations reduced activity to some extent, although not severely.

[0387] [Table 11]

[0388] One hybridoma clone, BLW-2E6, had a framework mutation Engineering resulted in 80 mutant clones, some of which are shown in Table 10. Eighty mutant clones were assayed for binding to primary human T cells (Figures 7 and 8 Count the T cells and dilute to 1 x 10^6 cells / mL, adding 5 μL of PBS per 100 μL of cells. Fc block (Human TruStain Fc Block, Biolegend) , 422302) and 0.5 μg / mL Live / Dead Filtration per 100 μL of cells xable Green Dead Cell Stain(Life Technol The cells were then incubated with 100 μL / well of PBS (Biogies, L-2301). Cells were cultured in a 96-well U-bottom plate (Falcon 3530) at 1 x 10^5 cells / well. The plate was centrifuged at 300 x g for 5 minutes to pellet the cells, and the supernatant was The supernatant was removed. The plate was gently vortexed briefly to resuspend the cells. The bryozoan supernatant was stained with FACS staining buffer (BSA, BD Biosciences 55 4657) at 7.5 μg / mL, 1.5 μg / mL, 0.3 μg / mL, and 0.06 μ 50 μL of each sample was added to the T cells and incubated at 4°C for 1 hour. Cells were washed once with staining buffer and stained with 5 μg / mL of secondary AF647 goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Catalog 10 50 μL of 9-605-097 was added to the cells. The plate was incubated at 4°C for 45 minutes. Wash the plate twice with staining buffer and twice with running buffer (staining buffer + 1 mM EDTA ( Life Technologies, AM9260G) + 0.1% Pluronic F-6 The plate was washed once with 8 (Life Technologies 24040-032). The cells were resuspended in 25 μL of running buffer and run on the HTFC screening system ( The results were analyzed using a PCR system (IntelliCyt Corporation). This indicates that the LC changes in the ribosomal region abolished binding, and therefore the mutations were not carried over. A slightly reduced binding was observed in the HC (CD3H231) in which all positions were reverted to the germline. Ta.

[0389] 1-7 BLW-2E6 LC C91 scanning Clone 2E6 and its derivatives showed poor expression and protein aggregation. One residue in the chain, C91, is predicted to be at risk for post-translational modification (PTM) and is involved in the protein All other possible amino acids were mutated to improve quality stability (Table 10). The V region DNA sequences were synthesized and cloned into the same human λ expression vector as the parent construct. The SPR results showed that a change to valine or leucine at position 91 significantly increased the binding This change was also incorporated into the wild-type sequence and was shown to not dramatically alter affinity. As a result of the above framework adaptation, antibody CD3B376 (CD3H219 / CD3L150) and CD3B450 (CD3H231 / CD3L197). and CD3B450 were cloned as IgG4PAA (S228P, F234A, IgG4 with the L235A mutation).

[0390] The sequence information for CD3B376 is provided below: CD3H219 HC amino acid sequence (SEQ ID NO: 640): QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIR QSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRN QFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK

[0391] CD3H219 HC nucleic acid sequence (SEQ ID NO: 712) caggtgcagctgcagcagtctggccctagactcgtgcgg ccttcccagaccctgtctctgacctgtgccatctccggcg actccgtgttcaacaacaacgccgcctggtcctggatccg gcagagcccttctagaggcctggaatggctgggccggacc tactaccggtccaagtggctgtacgactacgccgtgtccg tgaagtcccggatcaccgtgaaccctgacacctcccggaa ccagttcaccctgcagctgaactccgtgacccctgaggac accgccctgtactactgcgccagaggctactcctcctcct tcgactattggggccagggcaccctcgtgaccgtgtcctc t

[0392] CD3L150 LC amino acid sequence (SEQ ID NO: 676): QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQ HPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGL QAEDQADYHCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS

[0393] CD3L150 LC nucleic acid sequence (SEQ ID NO: 713) cagtctgctctgacccagcctgcctccgtgtctggctct cccggccagtccatcaccatcagctgtaccggcacctcct ccaacatcggcacctacaagttcgtgtcctggtatcagca gcaccccgacaaggcccccaaagtgctgctgtacgaggtg tccaagcggccctctggcgtgtcctccagattctccggct ccaagtctggcaacaccgcctccctgaccatcagcggact gcaggctgaggaccaggccgactaccactgtgtgtcctac gctggctctggcaccctgctgtttggcggaggcaccaagc tgaccgtgctg

[0394] VH amino acid sequence of CD3H219 (SEQ ID NO:652): qvqlqqsgprlvrpsqtlsltcaisgdsvfnnnaawswir qspsrglewlgrtyyrskwlydyavsvksritvnpdtsrn qftlqlnsvtpedtalyycargysssfdywgqgtlvtvss

[0395] VL amino acid sequence of CD3L150 (SEQ ID NO:661): qsaltqpasvsgspgqsitisctgtssnigtykfvswyqq hpdkapkvllyevskrpsgvssrfsgsksgntasltisgl qaedqadyhcVsyagsgtllfgggtkltvl

[0396] HCDR1 of CD3H219 (SEQ ID NO: 662): NNNAAWS

[0397] HCDR2 of CD3H219 (SEQ ID NO: 663): RTYYRSKWLYDYAVSV KS

[0398] HCDR3 of CD3H219 (SEQ ID NO: 664): GYSSSFDY

[0399] LCDR1 of CD3L150 (SEQ ID NO: 671): TGTSSNIGTYKFVS

[0400] LCDR2 of CD3L150 (SEQ ID NO: 673): EVSKRPS

[0401] LCDR3 of CD3L150 (SEQ ID NO: 690): VSYAGSGTLL

[0402] The sequence information for CD3B450 is provided below: CD3H231 HC amino acid sequence (SEQ ID NO: 675): QVQLQQSGPGLVKPSQTLSLTCAISGDSVFNNNAAWSWIR QSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITINPDTSKN QFSLQLNSVTPEDTAVYYCARGYSSSFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK

[0403] CD3H231 HC nucleic acid sequence (SEQ ID NO:714): caggtgcagctgcagcagagcggccccggcctggtcaagc ccagccagaccctgagcctgacctgcgccatcagcggcga cagcgtgttcaacaacaacgccgcctggtcctggatccgc cagagccccagccgcggcctggagtggctgggccgcacct actaccgcagcaagtggctgtacgactacgccgtgtccgt gaagtcccgcatcaccatcaaccccgacaccagcaagaac cagttctccctgcagctgaacagcgtgacccccgaggaca ccgccgtgtactactgcgcccgcggctacagcagcagctt cgactactggggccagggcaccctggtcaccgtgtccagc

[0404] CD3L197 LC amino acid sequence (SEQ ID NO:677): QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQ HPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTASLTISGL QAEDEADYYCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS

[0405] CD3L197 LC nucleic acid sequence (SEQ ID NO:715): Cagtctgctctgacccagcctgcctccgtgtctggctct cccggccagtccatcaccatcagctgtaccggcacctcct ccaacatcggcacctacaagttcgtgtcctggtatcagca gcaccccggcaaggcccccaaagtgatgatctacgaggtg tccaagcggccctccggcgtgtccaacagattctccggct ccaagtccggcaacaccgcctccctgacaatcagcggact gcaggccgaggacgaggccgactactactgtgtgtcctac gccggctctggcaccctgctgtttggcggcggaacaaagc tgaccgtgctg

[0406] VH amino acid sequence of CD3H231 (SEQ ID NO: 657): qvqlqqsgpglvk psqtlsltcaisgdsvfnnnaawswirqspsrglewlgrt yyrskwlydyavsvksritinpdtsknqfslqlnsvtped tavyycargysssfdywgqgtlvtvss

[0407] VL amino acid sequence of CD3L197 (SEQ ID NO:678): QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQ QHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTASLTISG LQAEDEADYYCVSYAGSGTLLFGGGTKLTVL

[0408] HCDR1 of CD3H231 (SEQ ID NO: 662): NNNAAWS

[0409] HCDR2 of CD3H231 (SEQ ID NO: 663): RTYYRSKWLYDYAVSV KS

[0410] HCDR3 of CD3H231 (SEQ ID NO: 664): GYSSSFDY

[0411] LCDR1 of CD3L197 (SEQ ID NO: 671): TGTSSNIGTYKFVS

[0412] LCDR2 of CD3L197 (SEQ ID NO: 673): EVSKRPS

[0413] LCDR3 of CD3L197 (SEQ ID NO: 690): VSYAGSGTLL

[0414] [Table 12]

[0415] 1-8 BLW-2 for hCD3ε construct by ProteOn SPR E6 CD3 mAb binding Recombinant human CD3ε(1-27) containing a C-terminal Tencon25 fusion (Jansse n production, referred to as hCD3ε(1-27)-Tn25) Binding of the BLW-2E6 anti-CD3 mAb with point mutations in the light and / or heavy chains was measured using Prot Measurement was performed using eOn SPR (Bio-Rad). The GLC Sensor Chip (Bio-Ra) was loaded with PBS containing 1000 kJ / ml at a flow rate of 30 μL / min. d, Catalog No. 176-5011) to all six ligand channels oriented perpendicular to the via amine coupling at 30 μg / mL in acetate buffer, pH 5.0 Goat anti-human Fc IgG (Jackson Immunoresearch, catalog no. The immobilization density was approximately 6000 response units on average. The variability between different channels was less than 5%. At 0.5 μg / mL (1000-1250 RU), five different antibodies were detected on the surface of anti-human Fc IgG. The sixth channel was a no-ligand surface control. hCD3ε(1-27)-Tn25 was used as an analyte at a concentration of 1 μM in a 3-fold dilution series of two concentrations. The captured mAb was bound to the mAb in a horizontal orientation. A sixth buffer sample was prepared to monitor separation and baseline stability. For all concentrations of hCD3ε(1-27)-Tn25, a flow rate of 100 μL / min was used. The dissociation phase was monitored for 30 min at RT. An 18 sec pulse was used to remove antigen and bound mAb. The binding surface was regenerated for the next interaction cycle with 0.8% phosphoric acid. The raw data were processed by subtracting two sets of baseline data from the data: 1) Interspot signal to correct for nonspecific interactions between Ag and the empty chip surface 2) to correct for baseline drift due to surface dissociation of captured mAb over time. Buffer channel signal for each mAb. Data processed at all concentrations were analyzed by 1: A global fit to a simple Langmuir binding model of 1 yielded the reaction rate constant (k on , k off ) and affinity (KD ) constant estimates were extracted. The results are given in Table 11.

[0416] [Table 13] N / A=Not Applicable

[0417] 1-9 Binding of anti-CD3 monoclonal antibodies to human T cells The in vitro binding affinities of CD3B376 and CD3B450 to human T cells were determined using antibodies Anti-CD3 transfection was assessed by flow cytometry after crossing with the original specific target arm. Preliminary studies were performed on human T cells to determine the saturation binding constant of the enzyme molecule (KdT). The immobilized mAb was then subjected to competitive binding assays using titrated concentrations of test mAb. A tracer ([T]) of a given concentration was used. The IC50 (the point at which 50% inhibition is achieved) of the test molecule was calculated. Using the K values, the following equation is used: d =IC50 / (1+([T] / K d Using T)) The binding affinity (K d ) was determined using five human donors. exaFluor488 SP34-2 anti-CD3 (BioScience#557705 ) saturation binding constant (K d T) was calculated (data not shown).

[0418] The saturation binding constant (K d T) decision Methods: Human pan-T cells were cryopreserved in a nitrogen tank until use. T cells were thawed and Wash with PBS, resuspend in FACS staining buffer, count (noting viability), and Resuspended at 0.5×10^6 cells / mL. Far Red Live / Dead st ain(Life Technologies, AKA Invitrogen #L3 4974) (50 μL of DMSO in a vial) was added at 1 μL per 1 × 10^6 cells. ;FcR blocker (Miltenyi Biotec #130-059-901) (0 1 mL of 1:20 dilution per 0.5 x 10^6 cells was added to the cells for 10 minutes. Cells were plated at 50,000 cells / well and washed. exaFluor488 SP-34 anti-CD3 was added to T cells for 2 hours at 4°C. The cells were washed to remove unbound antibody, fixed for 15 min, washed, and fixed with 1 mM EDTA. The cells were resuspended in FACS staining buffer containing

[0419] Binding was measured using an iQue Intellicyte Flow Cytometer Cells were gated for T cell populations, then cell individual fragments, and then viable cells (FL4). The geometric mean of staining (FL1) was determined for each well.

[0420] The resulting mean fluorescence intensity values ​​were plotted as a function of antibody molecule concentration and analyzed for single-site binding analysis (total binding). The Prism software was used to analyze the binding (Figure 9). Explaining the binding of antibody molecules to their receptors (CD3 on human pan-T cells) according to the law of mass action , the corresponding K d The formula is as follows: Y = (B max ×X) / (K d + X); (where Bmax is the maximum binding and K d is the linkage required to reach half-maximal binding. (The concentration of ATP is the concentration of ATP.)

[0421] Results: K for each donor d The values ​​were derived and the average values ​​were obtained. Coupling constant (Kd T) was derived to be 5.6 ± 1.0 nM (n = 4), and K d binding affinity was used in the formula already given to find

[0422] Determination of binding affinity of anti-CD3 mAb by competition assay Methods: Competitive binding assays were performed using bivalent antibodies against CD3. Anti-CD3 bivalent: CD3B376 and CD3B450 (Figure 10) Human pan-T cells were used to determine the binding affinity of the test mAbs. The tracer used was: Commercially available AlexaFluor 488 SP-34 anti-CD3 (BioScience #55 7705), and the saturation binding constant of this tracer is given above.

[0423] T cells were cryopreserved in a nitrogen tank until use. T cells were thawed and washed with PBS. The cells were then resuspended in FACS staining buffer and counted, taking note of viability, to obtain 0.5 x 10 6 cell Far Red Live / Dead stain (Live Technologies, AKA Invitrogen # L34974) Add 50 μL of DMSO to the culture at 1 μL per 1 x 10 cells; FcR blocker -(Miltenyi Biotec#130-059-901)(0.5×10^6A 1 mL of a 1:20 dilution each was added to the cells for 10 minutes. ,000 cells / well and washed.

[0424] mAb (and isotype control) were incubated at a starting concentration of 1000 or 200 μg / mL (2×). 1:2 serial dilutions from and mixed together at a fixed concentration (5 μg / mL; 2×) of tracer. , resulting in a 1x concentration. Therefore, the final concentration of the tracer (1x) is 2.5µg / mL = The mixture was added to the T cells for 2 hours at 4°C. The cells were washed to remove unbound antibody, fixed for 15 minutes, washed, and then incubated with 1 mM EDTA. The cells were resuspended in FACS staining buffer.

[0425] Binding was measured using an iQue Intellicyte Flow Cytometer Cells were gated for T cell populations, then cell individual fragments, and then viable cells (FL4). The geometric mean of the staining (FL1) was determined for each well. The mean fluorescence Intensity values ​​were plotted as a function of log antibody molecule concentration (converted to nM) and then calculated as EC50 / IC50 values ​​(nM) were derived from Prism in a sigmoidal dose response (variable slope) The analysis was performed using the following equation: d =IC50 / (1+([T] / K d T )) to calculate the binding affinity (K d ) was derived. d is the competitor (unlabeled molecule) is the affinity, IC50 of the test compound in nM, and [T] is the concentration of the tracer ( 16.6 nM) and K d T is the K of the tracer determined by saturation binding d is (5.6 nM for humans).

[0426] Production of monoclonal and bispecific antibodies The bispecific CD3 antibodies of the present invention are disclosed in Labrijn et al., 2013, PNA S, vol.110(13):5145-5150;International Publication No. 2011 / 131746 No.; or Labrijn et al., 2014, Nat Protoc, 9(10) :2450-63, generated by controlled Fab arm exchange (FAE). Briefly, this in vitro method involves providing two full-length parent bivalent antibodies, Each contains mutations in the antibody CH3 region that favor heterodimer formation, resulting in Bispecific antibodies containing half-arms derived from different parent antibodies are obtained. Mutations that can be used to advantage include, in the case of IgG1 antibodies, F40 in one parent antibody, 5L, R409K in the other parent antibody, or wild-type CH3 in the case of an IgG4 antibody. While retaining the F405L and K409R residues in one parent antibody.

[0427] The monospecific antibodies were expressed in HEK cell lines under the CMV promoter.

[0428] The parent antibody was eluted in an elution buffer of 100 mM NaAc pH 3.5, as well as 2 M Tris A Protein A column with a neutralization buffer of pH 7.5 and 150 mM NaCl was used. The mab was desalted using a PD10 (Sephadex G25M) column. The mixture was dialyzed against D-PBS, pH 7.2 buffer.

[0429] After purification, the parent antibody was mixed under reducing conditions in 75 mM cysteamine-HCl and incubated at 31°C. The recombination reaction was based on a molar ratio, with a set amount of target parent ( For example, 10 mg or about 71.8 nmoles) to a CD3 antibody (e.g., about 67.8 nmoles) ) and the targeting parent antibody was added at a 6% excess over the CD3 antibody. The recombinant product was then The reducing agent was removed by dialysis against BS. To minimize dosage, bispecific antibody reactions were performed using an excess of target parent antibody (ratio). Following partial reduction of the parent mAb, remove the reducing agent by dialysis overnight into PBS. Ta.

[0430] Results: Figure 10 shows the results of the AlexaFluor488 SP-34 anti-CD3 tracer antibody. Bivalent and monovalent anti-CD3 constructs, CD3B3, for competition for binding to Inhibition curves of Alexa 76 and CD3B450 are shown. Increasing concentrations of the tested anti-CD3 antibodies were Fluor488 tracer reduces antibody binding and therefore reduces mean fluorescence intensity (MFI ) to generate IC50 values, using the formula above, K d Affinity calculations, Table 1 I have summarized it in 3.

[0431] The CD3B376 binding site is tighter than CD3B450 in both bivalent and monovalent forms. there were.

[0432] [Table 14]

[0433] 1-10 Conformational stability of anti-CD3 monoclonal antibodies Anti-CD3 antibodies CD3B376 and CD3B389 (IgG1σ version of CD3B376) heavy chain is SEQ ID NO: 729; light chain is SEQ ID NO: 676), CD3B450, and and CD3B467 (IgG1σ version of CD3B450; heavy chain is SEQ ID NO: 728) The conformational stability of the nucleotide sequence ... The midpoint of the thermal transition, Tm, was determined from the thermal denaturation profile of each Ab candidate. Figures 12A-12E show the thermal denaturation profiles of anti-CD3 antibodies in PBS. Table 14 Regarding the thermal unfolding of anti-CD3 antibodies when determined by DSC It includes a summary of Tm and enthalpy values (ΔH).

[0434] The DSC results indicate that all anti-CD3 antibodies CD3B376, CD3B389, CD3B450 , and CD3B467 have folded domains. Based on the onset of the unfolding curve, the relative stability of each antibody was as follows: CD3B389 < C D3B467 < CD3B376 < CD3B450. The anti-CD3 molecules tested by DSC showed some differences in their thermal stability. The CD3B376 (IgG4 PAA) molecule showed three partially unresolved transitions at 59.7, 62.4, and 69.2 °C, and the total enthalpy of the unfolding was 417.6 kcal / mol, while the CD3B4 50 (IgG4 PAA) molecule showed two unresolved transitions at 62.5 and 66.3 °C, and the total enthalpy of the unfolding was 545.1 kcal / mol. The CD3 B389 (IgG1σ) molecule showed four transitions at 54.6, 58.2, 73.1, and 77.1 °C, and the total enthalpy of the unfolding was 401.7 kcal / mol, while the CD3B467 (IgG1σ) molecule showed four transitions at 56.3, 59.6, 66.5, and 75.6 °C, and the total enthalpy of the unfolding was 406.2 k cal / mol.

[0435]

Table 15

[0436] The two IgG1 antibodies showed a lower melting temperature (Tm) compared to the corresponding IgG4 PAA antibodies (having the same variable domains). ​The Tm of the first transition was 5-6°C lower (Figure 13). A and Figure 13B and Table 14).

[0437] 1-11 Binding of CD3B334 Fab in complex with the N-terminal peptide of CD3e crystal structure Anti-CD3 mAb CD3B334 (CD3H231 / CD3L137) was modified to By replacing many framework residues in the antibody with human germline residues, This procedure resulted in antibody CD3B334 with the following mutations: D43G / L49M / L in VL when compared to parental VL CD3L124 50I / S62N / Q85E / H89Y, VH R10G / R13k / V73I / R79K / T83S / L96V. CD3B33 The His-tagged Fab fragments of 4 were expressed in HEK293 Expi cells and Purification was performed using affinity chromatography and size exclusion chromatography. A 9-mer peptide from the N-terminus of CD3e was synthesized by New England Peptide ( It was synthesized using a Fab fragment (V1108-19 / 21) and mixed with Fab at a molar ratio of 10:1 (peptide excess). The vapor was extracted from a solution containing 4 M sodium formate in 0.1 M Tris, pH 8.5. The complex was crystallized by the diffusion method. The crystals were 66.5 × 69.4 × 100.4 Å in size. in the orthorhombic space group P212121 with lattice dimensions and one complex molecule in the asymmetric unit. Using the crystal structure of Fab as a search model, a resolution of 1.8 Å was obtained by molecular replacement. The structure of this complex was determined at high resolution.

[0438] CD3B334 bound to residues 1 to 6 of CD3e. The N-terminal Gln of the peptide In the hydrophobic environment, F107 of HCDR3 and L108 of LCDR3 are in the hydroxylated glutamate form. The two arginine residues R52 and R59 from HCDR2 were 6 associates with the acidic residues of CD3 through electrostatic interactions. In total, 16 residues are involved in the CD3B33 Residues from all CDRs except LCDR2 were found to form four paratopes. was in direct contact with (within 4 Å distance) (see Figure 19).

[0439] 2 PSMA antibody 2-1 Generation of PSMA cell lines Using standard molecular biology techniques, an in-house expression vector containing a CMV promoter was generated. to be used as a screening tool to evaluate anti-PSMA leads. To achieve this, full-length chimpanzee PSMA (H2Q3K5_PANTR, SEQ ID NO: 49) or full-length chimpanzee PSMA (H2Q3K5_PANTR, SEQ ID NO: 49) was used. An expression vector displaying full-length cynomolgus monkey PSMA (EHH56646.1, SEQ ID NO: 50) was prepared. The vector was transfected into HEK293F cells in suspension using standard methods. Transiently transfected 293F suspension cells were grown in a 200-well plate. When plated in medium plus serum, they became adherent and showed stable plasmid integration. Single cell populations were selected by serial dilution (as isotype controls) R-PE anti-rabbit secondary antibody (catalog number #111-116-144, Jackson ImmunoResearch Laboratories, Inc. and Usagipo Reclonal IgG (Cat. No. SC-532, Santa Cruz Biotec The primary antibody was PSMAL antibody (Center) affinity purified with chromosome (PSMAL). Heron polyclonal antibody (Cat. No. OAAB02483, Aviva System) PSMA surface receptor expression was quantified by FACS using the ELISA kit (Biology). .

[0440] Full-length human PSMA (FOLH1_HUMAN, SEQ ID NO: 51) and PSMA-positive cells Puromycin-containing lentivirus (Genecopoeia (Cat. No. EX-G0050-Lv105-10) was used to HEK293F cells (ATCC), which are PSMA negative, were transfected with lenalidomide. The cells were transduced with human HIV particles to overexpress human PSMA. By treating the cells, cells positively expressing PSMA and resistance markers were identified. Select and grow in DMEM + 10% HI FBS (Life Technologies) and supplemented with various concentrations of puromycin (Life Technologies). did.

[0441] In addition to HEK-generated cell lines, several commercially available cell lines were used for phage panning and binding and LNCaP clone FGC cells (ATCC catalog no. CRL-1740) is a commercially available human prostate cancer cell line. C4-2B cells were originally developed by MD Developed at Anderson, the LNs were grown in vivo and metastasized to the bone marrow. CaP is derived from FGCs (Thalmann, et al. 1994, Cancer Research 54,2577-81).

[0442] 2-2 Generation of soluble PSMA ECD protein Recombinant chimpanzee PSMA extracellular domain (ECD) protein (Chimp PSMA) The MA ECD (SEQ ID NO: 52) was generated for panning and purified using standard molecular biology techniques. We used an in-house expression vector containing a CMV promoter to generate an anti-PSMA lead. The N-terminal signal sequence (SEQ ID NO: 594), the N-terminal Avi tag (SEQ ID NO: 595) ), and the chimpanzee PSMA ECD gene containing a 6-His tag (SEQ ID NO: 596) The fragment (amino acids 44-750 of SEQ ID NO: 49) was isolated using standard molecular biology techniques. Cloned using an in-house expression vector containing a CMV promoter, 293Ex The gene was transiently expressed in pi cells (Invitrogen). DNA was prepared (U.S. Patent No. 6,670,127, U.S. Patent No. 6,521,427 The supernatant was collected and clarified by centrifugation. The protein was purified by 1) HisTrap HP 1) IMAC purification using a column (GE Healthcare), and 2) size exclusion purification. (Superdex200, Ge Healthcare) two-step purification process The elution buffer used here was 0.5 mM to stabilize PSMA dimerization. Dulbecco's Phosphate Buffered Saline with 1 M ZnCl2, Calcium, Magnesium (Thermofisher, #14040). Fraction containing the target protein The material was pooled and the protein concentration was determined by A280. This is used for the measurement of β-glucan and is called PSMG8.

[0443] Chimpanzee PSMA ECD was also biotinylated for panning. It contains an Avi tag. BirA promoter is co-transfected into mammalian cells to biotinylate target proteins. The plasmid was generated in-house. The BirA coding region (SEQ ID NO: 597) was cloned into mouse fused to the signal peptide from the IgG heavy chain (SEQ ID NO: 598) and the ER retention signal ( KDEL (SEQ ID NO: 716)) was added to the C-terminus to form the BirA plasmid (SEQ ID NO: 59 The constructed gene was cloned into an expression vector under the control of a CMV promoter. To produce biotinylated PSMA antigen, PSMA plasmid DNA was cloned. to the transfection mix in a 4-fold excess (w / w) over the BirA plasmid. Added.

[0444] Co-transfection of BirA expression constructs allows transcription of the chromosome via the Avi tag. Biotinylation of pansy PSMA ECD protein was performed, and the resulting secreted protein was 1) IMAC purification using a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex200, Ge Healthcare). The elution buffer was used to elute PSMA dimers. Dulbecco's phosphate buffered saline containing 0.5 mM ZnCl2 to stabilize , calcium, magnesium (Thermofisher, #14040). Proteins were tested for endotoxin before use in phage panning studies.

[0445] N-terminal signal (SEQ ID NO: 594), N-terminal Avi- (SEQ ID NO: 595) and 6His - (SEQ ID NO: 596) Recombinant corresponding to amino acids 44 to 750 of SEQ ID NO: 50, including the tag Cynomolgus monkey PSMA extracellular domain (ECD) protein (cynomolgus monkey PSMA E The chimpanzee PSMA ECD (CD, SEQ ID NO: 53) was cloned and analyzed as previously described for chimpanzee PSMA ECD. Cotransfection of the BirA expression construct resulted in the expression of Av Biotinylation of cynomolgus monkey PSMA ECD protein was performed via i-tag. The secreted proteins were purified using an IMAC HisTrap HP column (GE Healthcare) The purified product was purified by two-step purification using a MonoAvidin column. The protein was tested for endotoxin before use in phage panning studies. The material was also used for binding and affinity measurements and is designated PSMG1.

[0446] A second recombinant cyno PSMA ECD comprising IgG1 Fc (SEQ ID NO: 593) The protein (cynomolgus monkey PSMA Fc, SEQ ID NO: 54) was cloned and purified as a standard Expression using an in-house expression vector containing the CMV promoter using molecular biology techniques Cyno PSMA Fc protein was transiently transfected into 293HEK-expi cells. Transient transfection of PSMG3 in HEK293 Expi cells The cultures were harvested 5 days after transfection and centrifuged (30 min, 6000 rpm). The solution was clarified by filtration (0.2 μPES membrane, Corning) and filtered. Purified known IgG (same isoform) spiked into spent medium to The relative amount of IgG was determined using an Octet instrument (ForteBio).

[0447] The clarified cynomolgus monkey PSMA Fc supernatant was diluted with equilibrated (PBS, pH 7.2 ) HiTrap MabSelect Sure Protein A column (GE H Resin was loaded onto the PET / HEALTHCARE (Healthcare) at a relative concentration of approximately 30 mg of protein per mL of resin. After loading, the column was washed with dPBS, pH 7.2, and 10 column volumes of 0 The peak fractions were pooled and eluted with 2M Tris. The neutralized protein sample was diluted with Ca2+, Three modifications of dPBS containing Mg2+ and 0.5 mM ZnCl2, pH 7.2 The sample was dialyzed against 0.2 μL of HCl overnight at 4° C. The next day, the sample was removed from dialysis and filtered (0.2 μL). ), and analyzed by absorbance at 280 nm on a BioTek SynergyHT™ spectrophotometer. The amount of purified protein was determined by SDS-PAGE and analytical size analysis. The results were evaluated by exclusion HPLC (Dionex HPLC system). Pyrotell-T, Associates of Cape Cod) The purified protein was stored at 4°C.

[0448] Amino acid 4 of SEQ ID NO: 51 containing an N-terminal Avi- and 6His-tag (SEQ ID NO: 596) Recombinant human PSMA extracellular domain (ECD) protein (human PSMA) corresponding to 4-750 SMA ECD, SEQ ID NO: 55) and P The SMA ECD protein was expressed and purified as previously described.

[0449] 2-3 Identification of anti-chimpanzee and anti-human PSMA Fabs Panning with recombinant proteins Paired with four human VL germline gene (A27, B3, L6, O12) libraries De novo human Fab-p consisting of VH1-69, 3-23, and 5-51 heavy chain libraries IX library [Shi, L., et al. J Mol Biol, 2010.397 (2):pp.385-396. International Publication No. 2009 / 085462] First Solution Panic The cells were coated with biotinylated chimpanzee PSMA ECD according to the manufacturer's protocol. Streptavidin beads (Invitrogen catalog no. 112.0 5D, lot no. 62992920) and then following the manufacturer's protocol ProtG beads coated with cynomolgus monkey PSMA-Fc according to Invi Phages were captured on a phagocyte-specific antibody (Progen, Cat. No. 10003D) and subsequently analyzed by the manufacturer's protocol. Sera coated with biotinylated chimpanzee PSMA ECD according to protocol. -mag Double Speed ​​Magnetic Neutravidin Beads Phage capture on a phage filter (Thermo, Cat. No. 7815-2104-011150) This was performed using an alternating panning approach, which included one round of Two hits were obtained: PSMM18 and PSMM25.

[0450] Whole cell panning of anti-PSMAFab The chimpanzee ECD panning experiment described above or new de novo phages as input Using the round #1 output from the library, additional panning was performed on all cells. Briefly, phages were produced by helper phage infection, and the Concentration by PEG / NaCl precipitation according to standard protocols known in the art Untransfected parental HEK29 cells were incubated overnight at 4°C with gentle rocking. The phage library was pre-cleared on 3F cells. PEG / NaCl precipitation After incubation, the pre-depleted library was transfected into HEK293 cells expressing chimpanzee PSMA. The cells were incubated with either lysates or LNCAP cells for 2 hours at 4°C with gentle rocking. The removal of unbound phages and the recovery of phage-bound cells were performed using a Ficoll gradient. After several washing steps, the cells carrying the bound phage were incubated for 3 min without rocking. The mixture was incubated with 1 mL of TG-1 E. coli culture at 7°C for 30 minutes. The mixture was plated on LB-carbenicillin-1% glucose plates and incubated at 37°C. Growth was allowed to continue overnight, and the process was then repeated for subsequent panning rounds.

[0451] Conversion of phage Fab-pIX to Fab-His to generate E. coli supernatant The resulting phage Fab-pIX hits were converted to Fab-His using standard procedures. Plasmid DNA was then transferred to phage-panned E. coli (Plasmid Plus M). axi Kit, Qiagen catalog number 12963) and NheI / Spe The resulting 5400 and 100 bp fragments were then subjected to restriction enzyme digestion. The 5400 bp fragment was gel purified (MinElute PCR Purification Kit). The purified 5400 bp band was isolated using T4 Lipoprotein B12. The resulting product (Fab-his fusion) was self-ligated using gauze. The plasmid (encoding the nucleotide sequence) was transformed back into the TG-1 E. coli strain and clonally isolated. M Clone was isolated from the Fab-His clone by inducing the culture with IPTG overnight. After centrifugation of the overnight culture, the clarified supernatant was used in downstream assays. Preparation is complete. To determine the relative expression levels of the various Fab-his supernatants, serial dilutions are performed. Anti-kappa (Southern Biotech Catalog No. 2061-0 5) ELISA was performed. All clones tested showed similar Fab-his expression. (data not shown).

[0452] Cell binding of Fab-his fusions derived from Escherichia coli Evaluation of the binding ability of individual Fab-his fusions from E. coli supernatants to PSMA-expressing cells To investigate this, a cell-based binding assay was designed. All panning experiments after pIX excision Individual Fab clones were isolated from the round 3 output. In chimpanzee and cynomolgus monkey PSMA-expressing HEK cells and LNCaP cells Briefly, PSMA-expressing cells were cultured in 1000 x g of ... Dispense into V-bottom plates (CoStar 3357) at a density of 200,000 per well. Incubated with Fab fragment-expressing supernatant (100 μL) on ice for 1 hour. Wash the cells twice with PBS containing 2% FBS and incubate them on ice for 1 hour with mouse anti-human kappa antibody. -Stained with RPE antibody (Life Technologies Catalog No. MH10514) The cells were washed twice with PBS containing 2% FBS and then washed with 100 μL of the same wash buffer. Plates were read on a BD FACS array flow cytometer. Forward and side scatter were used to live gate the healthy population of cells, and then FACS data was analyzed by analyzing cells within this gate for PE staining. Analysis was performed using wJo software. Mean fluorescence intensity (MFI) was calculated and analyzed using Microsoft The data were exported to Excel. PSMA data from all three species (cynomolgus monkeys, chimpanzees, and human), and showed binding to the HEK293 cell line. Fab clones that showed no positive results were labeled as "preliminary positives." and carried forward for cloning into mammalian expression vectors for rescreening. True positives were selected from the binding of Fab supernatants expressed in mammalian cells to PSMA-expressing cell lines. did.

[0453] Preparation of mammalian Fab To convert the E. coli Fab into a mammalian-expressed Fab, follow the manufacturer's protocol. n-Fusion HD Cloning (ClonTech catalog number 638918) Briefly, the antibodies that passed the primary screening and were in mammalian Fab format were used. The nucleotide sequences of the transferred clones were analyzed using the InFu Primer Finder v 1.2.3 program (software developed in-house), which In-Fusion into huKappa_muIgGSP and huG1 Fab expression vectors isotype-specific fragments used to generate PCR fragments for sion cloning These vectors are based on pcDNA3.1. It is an in-house vector with a CMV promoter that does not allow for in-fusion processes. ...

Claims

[Claim 1] The invention described in the specification.

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