RU2812478C2 - ANTI-αvβ8 ANTIBODIES AND COMPOSITIONS AND THEIR USE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an isolated antibody or its antigen-binding fragment, which specifically bind α vβ 8 integrin, the method of its preparation, as well as compositions and kits containing it. An isolated nucleic acid that encodes the amino acid sequences of an antibody or an antigen-binding fragment thereof, as well as a vector and a cell containing it, are also disclosed. The invention also relates to a method of reducing activity of α vβ 8 integrin in the subject, as well as the method of determination ofα vβ 8 integrin in a sample, tissue or cell containing the above antibody or a fragment thereof.

EFFECT: invention is effective for treatment or prevention of cancer or a condition mediated by the activity ofα vβ 8 integrin.

51 cl, 31 dwg, 19 tbl, 26 ex

Description

This application claims priority to US Application No. 62/728,688, filed September 7, 2018, and US Application No. 62/890,945, filed August 23, 2019, the contents of which are incorporated herein by reference in their entirety.

LIST OF SEQUENCES

The description further incorporates by reference the Sequence Listing submitted September 5, 2019. In accordance with 37 C.F.R. § 1.52(e)(5), the Sequence Listing text file identified as PC72413A_SequenceListing_ST25.txt is 184,211 bytes in size and was created on September 3, 2019. The electronically filed Sequence Listing is within the scope of the description and therefore does not contain new entity.

SIDES OF JOINT RESEARCH

The invention currently claimed was created by or on behalf of the parties to the collaborative research agreement listed below. The collaborative research agreement entered into force on or before the date of creation of the claimed invention, and the claimed invention resulted from activities undertaken under the collaborative research agreement. The parties to the collaborative research agreement are THE REGENTS OF THE UNIVERSITY OF CALIFORNIA on behalf of its SAN FRANCISCO CAMPUS and PFIZER INC.

TECHNICAL FIELD

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind αvβ8 integrin, and their compositions, methods and uses.

BACKGROUND OF THE ART

Transforming growth factor β (TGFβ) is a potent suppressor of adaptive and innate immunity and an important mediator of immune suppression by a subset of regulatory T cells. TGFβ is required for the induction of Th17 cells, which can promote tumor progression through the induction of granulocytic inflammation and promotes epithelial-mesenchymal transformation of tumor cells, secretion and accumulation of fibrous tumor stroma, which may contribute to the exclusion of immune cells from some solid tumors. For all these reasons, TGFβ inhibition has been investigated as an adjunctive immunotherapy, especially in so-called “immune-excluded” tumors (Gorelik et al. Nat. Med. 7: 1118-1122, 2001; Tauriello et al. Nature 554:538-543, 2018; Mariathasan et al. Nature 554:544-548, 2018, Dodagatta et al. J Immunother Cancer. 7: 62. 2019; US Patent No. 10,167,334). However, since TGFβ plays important homeostatic roles in many biological systems, systemic targeting of TGFβ signaling poses many challenges due to unwanted side effects (Hata and Akhurst Nat. Rev. Drug Dev. 11, 791-811, 2012; Akhurst et al Cold Spring Harbor Perspectives 10, 2017; Flavell et al. Nat. Rev. Immunol. 10:554-567, 2010).

Previous studies have shown that inhibition of TGFβ signaling can enhance responses to radiation or vaccine therapy when combined with checkpoint inhibition (Vanpouille-Box et al. Cancer Res . 75:2232-2242, 2015; Terabe et al. OncoImmunology 6(5) :e1308616, 2017). In vivo , TGFβ activity is regulated through several mechanisms. For example, TGFβ is secreted as an inactive or latent complex, where the cleavage latency-associated peptide (LAP) domain comprises the active TGFβ mature peptide. Latent-TGFβ can be covalently associated with the extracellular matrix via latent TGFβ binding protein (LTBP) or expressed at the cell surface via glycoprotein-A repeat protein (GARP). Early in vitro data showed that the latent TGFβ complex can be activated by high temperature, acidic pH, and various proteases (Annes et al. J Cell Sci. 116:217–24, 2003), but the importance of these mechanisms in vivo remains to be determined.

The role of αv integrin family members, more specifically αvβ1, αvβ6 and αvβ8, has been demonstrated for the activation of latent-TGFβ. Integrin αvβ8 is a transmembrane noncovalent heterodimer consisting of ITGαV and ITGβ8 subunits. Expression of αvβ8 is unique among αv integrins, where its expression by immune cells such as dendritic cells, T regulatory cells and tumor-associated macrophages occurs as an ad hoc activator of TGFβ to regulate active immune responses. Expression of αvβ8 by dendritic cells (DCs) acts as a mediator of TGFβ production during T cell stimulation and strongly influences the differentiation and development of Tregs and Th17 cells through Th1 differentiation during immune responses. Mice with conditional deletion of Itgb8 in DCs or all leukocytes show potent inhibition of TGFβ-dependent induction of antigen-specific Th17 cells, and are subsequently protected from organ dysfunction in certain preclinical models of autoimmunity, such as multiple sclerosis (experimental autoimmune encephalomyelitis) and allergic asthma (Travis et al Nature 449(7160):361-5, 2007; Melton et al. J Clin Invest 120(12):4436-44, 2010).

TGFβ plays a role in both the differentiation and recruitment of suppressor immune cells into tumors and as a tumor intrinsic factor that contributes to an immunosuppressive tumor microenvironment. In some forms of cancer, TGFβ may promote tumor development by influencing multiple aspects of the tumor microenvironment, including angiogenesis, metastasis, epithelial-mesenchymal transition, and, perhaps most importantly, suppression of infiltrating immune cells.

Consequently, in view of the prominent role of TGFβ in the tumor microenvironment and the numerous challenges associated with systemic targeting of TGFβ signaling (Hata and Akhurst Nat. Rev. Drug Dev. 11, 791-811, 2012; Akhurst et al. Cold Spring Harbor Perspectives 10, 2017 (Flavell et al. Nat. Rev. Immunol . 10:554–567, 2010), there is a need to develop strategies to selectively inhibit αvβ8-dependent activation of latent TGFβ.

SUMMARY OF THE INVENTION

Disclosed herein are antibodies (e.g., humanized and chimeric antibodies) and antigen-binding fragments thereof that specifically bind to αvβ8 integrin (also interchangeably referred to herein as “AVB8,” “αvβ8,” or “avb8”) (e.g., human αvβ8 integrin , mouse, cynomolgus and/or rat). In certain aspects, antibodies and antigen-binding fragments thereof bind to αvβ8 integrin and significantly reduce TGFβ signaling (eg, TGFβ1 and TGFβ3), for example in a tumor or tumor microenvironment.

Mature TGFβ is present in an inactive or latent form in complex with the latency-associated peptide (LAP) domain. Binding of αvβ8 integrin to LAP releases active TGFβ (eg, TGFβ1 and TGFβ3). Reduced αvβ8 integrin binding to LAP may prevent the release of active TGFβ, thereby reducing TGFβ signaling. TGFβ is known to have immunosuppressive effects, for example, in the tumor microenvironment, so reducing TGFβ activity and/or signaling using the antibodies described herein may result in activation of an immune response, such as an antitumor response in vivo .

Due to the limited expression of αvβ8 integrin on immune cells (eg, dendritic cells, T regulatory cells, tumor-associated macrophages) and tumor cells, the antibodies described herein may provide a more targeted non-systemic reduction in TGFβ signaling. Thus, the antibodies and antigen-binding fragments thereof herein enable more selective antagonism of TGFβ activity in the immune system and/or tumor microenvironment, thereby improving the antitumor response in a subject. In some embodiments described herein, antibodies against αvβ8 integrin have been shown to cause growth suppression and/or complete tumor regression in animal models for several types of cancer, including, for example, squamous cell carcinoma, breast cancer and/or colon cancer , alone or in combination with other immunomodulators such as checkpoint inhibitor modulators (eg, PD-1, PD-L1, CTLA-4 inhibitors or 4-1BB agonists) anticancer therapies, such as radiotherapy.

Therefore, in certain aspects, the disclosure provides antibodies and antigen-binding fragments thereof that bind to αvβ8 integrin with high affinity and specificity, nucleic acid molecules encoding the antibodies and antigen-binding fragments thereof, expression vectors, host cells, and methods for producing them. In certain aspects, antibodies and antigen-binding fragments thereof exhibit altered effector functions (eg, have reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and/or reduced complement-dependent cytotoxicity (CDC)). In certain aspects, anti-αvβ8 integrin antibodies and their antigen binding fragments exhibit improved binding affinity for αvβ8 integrin compared to the mouse hybridoma antibodies and their antigen binding fragments from which they are derived. The humanized anti-αvβ8 integrin antibodies and antigen binding fragments thereof described herein may be used alone or in combination with other agents or therapeutic modalities (eg, immunomodulators or anticancer therapies) for the treatment, prevention and/or diagnosis of disorders, such as cancer disorders (for example, tumors of hard and soft tissues). Thus, compositions and methods for detecting αvβ8 integrin, as well as methods for treating various disorders, including cancer, using anti-αvβ8 integrin antibodies and antigen-binding fragments thereof are disclosed.

Those skilled in the art will understand or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following embodiments (E).

E1. An isolated antibody or antigen binding fragment thereof that specifically binds to αvβ8 integrin, wherein said antibody or antigen binding fragment thereof has at least one of the following properties:

i. binding affinity, expressed as KD, for human αvβ8 integrin, which is less than the KD for mouse ADWA11 antibody, as described in US Patent No. 9,969,804, which is incorporated herein by reference in its entirety, confirming the amino acid sequences and presented in, for example , SEQ ID NO: 20- 33 and 71-76 of the present specification, for example, wherein the ADWA11 antibodies of the invention have a KD of less than 536 pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 500, 510, 520, 530, 531, 532, 533, 534 or 535 pM);

ii. KD for human αvβ8 integrin that is less than or equal to 200 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 , 90, 100, 150, 180, 190 or 200 pM), for example, for purified human αvβ8 integrin;

iii. KD for human αvβ8 integrin, which is less than or equal to 100 pM for purified human αvβ8 integrin;

iv. A KD for a mouse αvβ8 integrin that is less than a KD for a mouse ADWA11 antibody, e.g., less than 489 pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 460, 470, 480, 485, 486, 487 or 488 pM);

v. KD for mouse αvβ8 integrin, which is 70.8 +/- 19.9 pM for purified mouse αvβ8 integrin;

vi. KD for cynomolgus αvβ8 integrin that is less than the KD for mouse ADWA11 antibody, e.g., less than 507 pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 , 200, 250, 300, 350, 370, 400, 450, 500, 501, 502, 503, 504, 505 or 506 pM);

vii. a KD for cynomolgus αvβ8 integrin that is less than or equal to 100 pM for purified cynomolgus αvβ8 integrin;

viii. KD for rat αvβ8 integrin, which is approximately 160 pM;

ix. approximately equivalent affinity for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, e.g., with a KD of less than 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98 pM), for example, determined using the Biacore affinity assay;

X. IC50 for inhibition of TGFβ transactivation that is less than that of the mouse ADWA11 antibody, e.g., less than 183 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 181 or 182 pM);

xi. IC50 for inhibition of TGFβ transactivation in U251 cells approximately 199 +/- 93.6 pM;

xii. IC50 for inhibition of TGFβ transactivation, about 100 pM to about 300 pM;

xii. EC50 for U251 cells approximately 126 +/- 34 pM (e.g. approximately 50, 60, 70 80, 90, 100, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 140, 150, 160, 170, 180 or 190 pM);

xiv. EC50 for U251 cells approximately 256 +/- 115 pM (eg, approximately 120, 140, 160, 180, 200, 220, 240, 260, 280, 290, 300, 320, 340, 360, 380, 400 pM);

xv. EC50 for U251 cells from about 80 pM to about 400 pM;

xvi. EC50 for C8-S cells is approximately 115 pM;

xvii. EC50 for C8-S cells approximately 145+/- 23.7 pM;

xviii. EC50 for C8-S cells from about 110 pM to about 180 pM;

xix. at least one predicted human pharmacokinetics (PK) parameter selected from:

A. clearance from the central chamber (CL) is approximately 0.12-0.15 ml/h/kg;

b. intercompartmental clearance of distribution (CLF) approximately 0.15-0.51 ml/h/kg;

With. volume of distribution for the central chamber (V1) is approximately 36-39 ml/kg;

d. volume of distribution for the peripheral chamber (V2) approximately 21 -33 ml/kg; and/or

e. the terminal half-life (t _1/2 ) is approximately 12 days;

f. terminal half-life (t _1/2 ) approximately 15-17 days; or

xx. lack of detectable binding to human Fcγ or C1q receptors.

E2. The isolated antibody or antigen binding fragment thereof, from embodiment E1, wherein the KD for human αvβ8 integrin is less than the KD for mouse ADWA11 antibody, e.g., less than 536 pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 500, 510, 520, 530, 531, 532, 533, 534 or 535 pM).

E3. An isolated antibody or antigen binding fragment thereof, from embodiment E1 or E2, wherein the KD for human αvβ8 integrin is less than or equal to 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 pM), for example, for purified human αvβ8 integrin.

E4. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the KD for mouse αvβ8 integrin is less than the KD for mouse ADWA11 antibody, e.g., less than 489 pM (e.g., 1, 5, 10, 20, 30, 40, 50 , 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 460, 470, 480, 485, 486, 487 or 488 pM).

E5. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the KD for mouse αvβ8 integrin is approximately 70.8 +/- 19.9 pM.

E6. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, wherein the KD for cynomolgus αvβ8 integrin is less than the KD for mouse ADWA11 antibody, e.g., less than 507 pM (e.g., 1, 5, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 500, 501, 502, 503, 504, 505 or 506 pM).

E7. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the KD for cynomolgus αvβ8 integrin is less than 100 pM.

E8. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the KD for rat αvβ8 integrin is approximately 160 pM.

E9. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, wherein the isolated antibody or antigen-binding fragment thereof shows approximately equivalent affinity for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, e.g., with KD less than 100 pM (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95 pM), e.g. , determined using the Biacore affinity assay.

E10. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, wherein the isolated antibody or antigen-binding fragment thereof shows approximately equivalent affinity for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, e.g., with KD less than 100 pM (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98 pM) , for example, determined using the Biacore affinity assay.

E11. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the IC50 for inhibiting TGFβ transactivation is less than that of the mouse ADWA11 antibody, e.g., less than 183 pM (e.g., 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 181 or 182 pM) .

E12. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the IC50 for inhibition of TGFβ transactivation in U251 cells is approximately 199 +/- 93.6 pM.

E13. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the IC50 for inhibiting TGFβ transactivation is from about 100 pM to about 300 pM.

E14. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the EC50 for U251 cells is approximately 126 pM with a standard deviation of plus/minus 34 pM.

E15. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the EC50 for U251 cells is approximately 256 pM with a standard deviation of plus/minus 115 pM.

E16. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the EC50 for U251 cells is from about 100 pM to about 400 pM.

E17. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the EC50 for C8-S cells is approximately 115 pM.

E18. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the EC50 for C8-S cells is approximately 145 +/- 23.7 pM.

E19. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the EC50 for C8-S cells is from about 110 pM to about 180 pM.

E20. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, having at least one predicted human pharmacokinetic (PK) parameter selected from the group consisting of:

(i) clearance from the central chamber (CL) is approximately 0.12-0.15 ml/h/kg;

(ii) intercompartmental clearance of distribution (CLF) of approximately 0.15-0.51 ml/h/kg;

(iii) volume of distribution for the central chamber (V1) approximately 36-39 ml/kg;

(iv) volume of distribution for the peripheral chamber (V2) approximately 21-33 ml/kg; and/or

(v) the terminal half-life (t _1/2 ) is approximately 12-17 days.

E21. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, wherein the isolated antibody or antigen-binding fragment thereof does not show detectable binding to the human Fcγ receptor or C1q.

E22. An isolated antibody or antigen-binding fragment thereof, according to any of the above embodiments, wherein the antibody or antigen-binding fragment thereof further has at least one of the following properties:

(i) binds specifically to αvβ8 integrin (eg, human, mouse, cynomolgus and/or rat αvβ8 integrin);

(ii) reduces the interaction between αvβ8 integrin and latency-associated peptide (LAP);

(iii) reduces TGF-β signaling;

(iv) effectively blocks αvβ8 integrin-mediated TGFβ activation with an IC50 ≤10 nM;

(v) has a comparable Kd (within 5-fold) to the non-human primate ortholog (NHP);

(vi) selectively binds human αvβ8 and does not detectably bind αvβ8 homolog (eg, αvβ1, αvβ3, αvβ5, and αvβ6);

(vii) induces growth suppression and/or complete tumor regression in an animal model of cancer, alone or in combination with an immunomodulatory agent, e.g., a checkpoint inhibitor modulator, e.g., PD-1, PD-L1, CTLA-4 inhibitors or a stimulatory molecule agonist , for example, 4-1BB;

(viii) causes suppression of growth and/or complete tumor regression in an animal model of cancer in combination with anticancer therapy, such as radiotherapy;

(ix) shows at least a 60% reduction in tumor growth in a syngeneic tumor xenograft model, e.g., when administered at a dose of ≤10 mg/kg, alone or in combination with an immunomodulatory agent (e.g., a PD-1, PD-L1, or CTLA inhibitor -4);

(x) enhances an antitumor response in the presence of one or more immunomodulators, e.g., a checkpoint inhibitor antagonist, e.g., a PD-1, PD-L1, or CTLA-4 antagonist, or an immune response activator, e.g., a 4-1BB agonist, when administered to a subject ;

(xi) has an efficacy that is independent of αvβ8 integrin expression in a tumor model;

(xii) increases the abundance of CD8+ GzmB+ T cells in the tumor microenvironment;

(xiii) shows a reduction, e.g., at least >80% reduction in tumor growth when used in combination with a checkpoint inhibitor antagonist (e.g., anti-PD-1 or anti-PD-L1 antibody), e.g., in a syngeneic model of squamous cell carcinoma, breast cancer and/or colon cancer;

(xiv) demonstrates a statistically significant improvement in overall subject survival by Kaplan-Meier analysis;

(xv) has a high degree of thermal stability;

(xvi) shows minimal aggregation at high concentrations; And

(xvii) can demonstrate reproducible expression and purity under industrial production conditions.

E23. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

one, two or three CDRs from the heavy chain variable region (eg, H1, H2 or H3) and/or one, two or three CDRs from the light chain variable region (eg, L1, L2 or L3) selected from:

(i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 8 or 14,

CDR-H2 containing the amino acid sequence SEQ ID NO: 9 or 15,

CDR-H3 containing the amino acid sequence SEQ ID NO: 10 or 16,

CDR-L1 containing the amino acid sequence SEQ ID NO: 11 or 17,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 12 or 18, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 13 or 19, or

(ii) CDR-H1 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 8 or 14,

CDR-H2 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 9 or 15,

CDR-H3 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 10 or 16,

CDR-L1 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 11 or 17,

CDR-L2 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 12 or 18 or

CDR-L3 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 13 or 19, optionally where:

any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3, not containing the amino acid sequence of any of:

(a) SEQ ID NO: 22, 23, 24, 25, 26 and 27, respectively,

(b) SEQ ID NO: 28, 29, 30, 31, 32 and 33, respectively,

(c) SEQ ID NO: 22, 23, 24, 71, 72 and 73, respectively, or

(d) SEQ ID NO: 28, 29, 30, 74, 75 and 76, respectively.

Alternatively or in combination with any of the embodiments presented herein (eg, E1-E23), an antibody or antigen binding fragment thereof having one or more of the following aspects, features and embodiments.

E24. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising:

one, two or three CDRs from the heavy chain variable region (eg, H1, H2 or H3), and/or one, two or three CDRs from the light chain variable region (eg, L1, L2 or L3) selected from:

(i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 8,

CDR-H2 containing the amino acid sequence SEQ ID NO: 9,

CDR-H3 containing the amino acid sequence SEQ ID NO: 10,

CDR-L1 containing the amino acid sequence SEQ ID NO: 11,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 12, and

CDR-L3 containing the amino acid sequence of SEQ ID NO: 13, or

(ii) a CDR-H1 comprising at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 8,

CDR-H2 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 9,

CDR-H3 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 10,

CDR-L1 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 11,

CDR-L2 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 12 or

CDR-L3 comprising at least one amino acid change, but not more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 13, optionally where:

any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 does not contain the amino acid sequence of any of:

(a) SEQ ID NO: 22, 23, 24, 25, 26 and 27, respectively, or

(b) SEQ ID NO: 22, 23, 24, 71, 72 and 73, respectively.

E25. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

one, two or three complementarity determining regions (CDRs) from the heavy chain variable region (eg, H1, H2 or H3) and/or one, two or three CDRs from the light chain variable region (eg, L1, L2 or L3) selected from:

CDR-H2 containing the amino acid sequence SEQ ID NO: 9,

CDR-L1 containing the amino acid sequence SEQ ID NO: 11,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 12, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 13.

E26. An isolated antibody or antigen binding fragment thereof from embodiment E24 or E25, comprising:

CDR-H1 containing the amino acid sequence SEQ ID NO: 8,

CDR-H2 containing the amino acid sequence of SEQ ID NO: 9, and

CDR-H3 containing the amino acid sequence SEQ ID NO: 10.

E27. An isolated antibody or antigen-binding fragment thereof, according to any one of embodiments E24-E26, comprising:

CDR-L1 containing the amino acid sequence SEQ ID NO: 11,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 12, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 13.

E28. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 8,

CDR-H2 containing the amino acid sequence SEQ ID NO: 9,

CDR-H3 containing the amino acid sequence SEQ ID NO: 10,

CDR-L1 containing the amino acid sequence SEQ ID NO: 11,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 12, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 13.

E29. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region (e.g., H1, H2, or H3) and/or one, two, or three CDRs from the light chain variable region (for example, L1, L2 or L3):

CDR-H1 containing the amino acid sequence DYYMN (SEQ ID NO: 8);

CDR-H2 containing the amino acid sequence WIDPD X ₁ GNTIY X ₂ PKFQG (SEQ ID NO: 131), where X ₁ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or Q; and X ₂ can be any of: an amino acid, an amino acid other than D, a conservative substitution of D, D or E;

CDR-H3 containing the amino acid sequence RLLMDY (SEQ ID NO: 10);

CDR-L1 containing the amino acid sequence RSTKSL X ₃ HFNGNTYLF (SEQ ID NO: 132), where X ₃ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or S;

CDR-L2 containing the amino acid sequence YYMS X ₄ LAS (SEQ ID NO: 133), where X ₄ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or S; and/or

CDR-L3 containing the amino acid sequence X ₅ QSLEYPFT (SEQ ID NO: 134), where X ₅ can be any of: an amino acid, an amino acid other than M, a conservative substitution M, M or Q;

for example, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences of SEQ ID NO: 22, 23, 24, 25, 26 and 27, respectively, or SEQ ID NO: 22, 23, 24, 71, 72 and 73, respectively.

E30. An isolated antibody or antigen binding fragment thereof, from embodiment E29, wherein X ₁ is Q and X ₂ is E.

E31. An isolated antibody or antigen binding fragment thereof, from embodiment E29 or E30, wherein _X3 is S.

E32. An isolated antibody or antigen binding fragment thereof, as in any one of embodiments E29-E31, wherein _X4 is S.

E33. An isolated antibody or antigen binding fragment thereof, as in any one of embodiments E29-E32, wherein _X5 is Q.

E34. An isolated antibody or antigen binding fragment thereof, from embodiment E29, wherein X ₁ is Q, X ₂ is E, X ₃ is S, and X ₅ is Q.

E35. An isolated antibody or antigen binding fragment thereof, from embodiment E29, wherein X ₁ is Q, X ₂ is E, and X ₃ is S.

E36. An isolated antibody or antigen binding fragment thereof, from embodiment E29, wherein X ₁ is Q, X ₂ is E, X ₃ is S, X ₄ is S, and X ₅ is Q.

E37. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region (e.g., H1, H2, or H3), and/or one, two, or three CDR from the light chain variable region (e.g. L1, L2 or L3):

CDR-H1 containing the amino acid sequence DYYMN (SEQ ID NO: 8);

CDR-H2 containing the amino acid sequence WIDPD X ₁ G X ₂ TIY X ₃ X ₄ X ₅ X ₆ X ₇ G (SEQ ID NO: 167), where X ₁ can be any of: an amino acid, an amino acid other than N, a conserved substitutions N, N or Q; X ₂ can be any of: an amino acid, an amino acid other than N, a conservative substitution for N, N, or Q; X ₃ can be any of: an amino acid, an amino acid other than D, a conservative substitution of D, D or E; X ₄ can be any of: an amino acid, an amino acid other than P, a conservative substitution P, P, Q, D or A; X ₅ can be any of: an amino acid, an amino acid other than K, a conservative substitution of K, K, S or A; X ₆ can be any of: an amino acid, an amino acid other than F, a conservative substitution of F, F or V; and X ₇ can be any of: an amino acid, an amino acid other than Q, a conservative substitution of Q, Q or K;

CDR-H3 containing the amino acid sequence RLLMDY (SEQ ID NO: 10);

CDR-L1 containing the amino acid sequence RSTKS X ₈ X ₉ HFNGN X ₁₀ YLF (SEQ ID NO: 168), where X ₈ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or I; X ₉ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or S; and X ₁₀ can be any of: an amino acid, an amino acid other than T, a conservative substitution T, T or S;

CDR-L2 containing the amino acid sequence Y X ₁₁ X ₁₂ S X ₁₃ L X ₁₄ S (SEQ ID NO: 169), where X ₁₁ can be any of: an amino acid, an amino acid other than Y, a conservative substitution of Y, Y or A ; X ₁₂ can be any of: an amino acid, an amino acid other than M, a conservative substitution of M, M or A; X ₁₃ can be any of: an amino acid, an amino acid other than N, a conservative substitution for N, N, or S; and X ₁₄ can be any of: an amino acid, an amino acid other than A, a conservative substitution of A, A or Q; and/or

CDR-L3 containing the amino acid sequence X ₁₅ QS X ₁₆ X ₁₇ X ₁₈ P X ₁₉ T (SEQ ID NO: 170), where X ₁₅ can be any of: an amino acid, an amino acid other than M, a conservative substitution M, M, or Q; X ₁₆ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or Y; X ₁₇ can be any of: an amino acid, an amino acid other than E, a conservative substitution of E, E or S; X ₁₈ can be any of: an amino acid, an amino acid other than Y, a conservative substitution of Y, Y or T; and X ₁₉ can be any of: an amino acid, an amino acid other than F, a conservative substitution F, F, L or W;

for example, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences of SEQ ID NO: 22, 23, 24, 25, 26 and 27, respectively, or SEQ ID NO: 22, 23, 24, 71, 72 and 73, respectively, optionally where:

X ₁ is Q, X ₂ is N, X ₃ is E, X ₄ is P, X ₅ is K, X ₆ is F and X ₇ is Q,

X ₈ is L, X ₉ is S and X ₁₀ is T,

X ₁₁ is Y, X ₁₂ is M, X ₁₃ is S and X ₁₄ is A, and/or

X ₁₅ is Q, X ₁₆ is L, X ₁₇ is E, X ₁₈ is Y and X ₁₉ is F.

E38. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region (e.g., H1, H2, or H3), and/or one, two, or three CDR from the light chain variable region (e.g. L1, L2 or L3):

CDR-H1 containing the amino acid sequence DYYMN (SEQ ID NO: 8);

CDR-H2 containing the amino acid sequence WIDPD X ₁ GNTIY X ₂ PK X ₃ QG (SEQ ID NO: 171), where X ₁ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or Q; X ₂ can be any of: an amino acid, an amino acid other than D, a conservative substitution of D, D or E; and X ₃ can be any of: an amino acid, an amino acid other than F, a conservative substitution of F, F or V;

CDR-H3 containing the amino acid sequence RLLMDY (SEQ ID NO: 10);

CDR-L1 containing the amino acid sequence RSTKSL X ₄ HFNGNTYLF (SEQ ID NO: 172), where X ₄ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or S;

CDR-L2 containing the amino acid sequence YY X ₅ S X ₆ LAS (SEQ ID NO: 173), where X ₅ can be any of: an amino acid, an amino acid other than M, a conservative substitution M, M or A; and X ₆ can be any of: an amino acid, an amino acid other than N, a conservative substitution for N, N, or S; and/or

CDR-L3 containing the amino acid sequence X ₇ QS X ₈ EYPFT (SEQ ID NO: 174), where X ₇ can be any of: an amino acid, an amino acid other than M, a conservative substitution M, M or Q; and X ₈ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or Y;

for example, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences of SEQ ID NO: 22, 23, 24, 25, 26 and 27, respectively, or SEQ ID NO: 22, 23, 24, 71, 72 and 73, respectively, optionally where:

X ₁ is Q, X ₂ is E and X ₃ is F,

X ₄ is S,

X ₅ is M and X ₆ is S, and/or

X ₇ is Q and X ₈ is L.

E39. An isolated antibody or antigen binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three of the CDR sequences from the VH region containing the amino acid sequence of SEQ ID NO: 6, wherein the CDR sequences are defined according to Kabat.

E40. An isolated antibody or antigen binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from the VL region containing the amino acid sequence of SEQ ID NO: 7, wherein the CDR sequences are defined according to Kabat.

E41. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from the VH region containing the amino acid sequence of SEQ ID NO: 6, and one, two or three CDR-L1 sequences from the VL region containing the amino acid sequence of SEQ ID NO:7, where the CDR sequences are determined according to Kabat.

E42. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from a polypeptide encoded by nucleic acid sequence SEQ ID NO: 189, 190 or 191, wherein the CDR sequences are defined according to Kabat.

E43. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from a polypeptide encoded by the nucleic acid sequence SEQ ID NO: 185 or 186, wherein the CDR sequences are defined according to Kabat.

E44. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from a polypeptide encoded by the nucleic acid sequence SEQ ID NO: 189, 190 or 191 and one, two or three CDR sequences from the polypeptide , encoded by the nucleic acid sequence SEQ ID NO: 185 or 186, where the CDR sequences are determined according to Kabat.

E45. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing one or more complementarity determining regions (CDRs), selected from:

(i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 14,

CDR-H2 containing the amino acid sequence SEQ ID NO: 15,

CDR-H3 containing the amino acid sequence SEQ ID NO: 16,

CDR-L1 containing the amino acid sequence SEQ ID NO: 17,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 18, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 19; or

(ii) a CDR-H1 comprising at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 14,

CDR-H2 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 15,

CDR-H3 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 16,

CDR-L1 containing at least one amino acid change, but no more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 17,

CDR-L2 containing at least one amino acid change, but no more than two, three or four changes (eg, substitution, deletion or insertion, such as a conservative substitution) relative to SEQ ID NO: 18, or

CDR-L3 comprising at least one amino acid change, but not more than two, three or four changes (e.g., substitution, deletion or insertion, e.g., conservative substitution) relative to SEQ ID NO: 19, optionally where:

any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 does not contain the amino acid sequences of any of

(a) SEQ ID NO: 28, 29, 30, 31, 32 and 33, respectively, or

(b) SEQ ID NO: 28, 29, 30, 74, 75 and 76, respectively.

E46. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing one or more complementarity determining regions (CDRs) selected from:

CDR-H2 containing the amino acid sequence SEQ ID NO: 15,

CDR-L1 containing the amino acid sequence of SEQ ID NO: 17, and

CDR-L2 containing the amino acid sequence SEQ ID NO: 18.

E47. An isolated antibody or antigen binding fragment thereof, from embodiment E45 or E46, comprising:

CDR-H1 containing the amino acid sequence SEQ ID NO: 14,

CDR-H2 containing the amino acid sequence of SEQ ID NO: 15, and

CDR-H3 containing the amino acid sequence SEQ ID NO: 16.

E48. An isolated antibody or antigen-binding fragment thereof, according to any one of embodiments E45-E47, comprising:

CDR-L1 containing the amino acid sequence SEQ ID NO: 17,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 18, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 19.

E49. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 14,

CDR-H2 containing the amino acid sequence SEQ ID NO: 15,

CDR-H3 containing the amino acid sequence SEQ ID NO: 16,

CDR-L1 containing the amino acid sequence SEQ ID NO: 17,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 18, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 19.

E50. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence GFNIKDYYMN (SEQ ID NO: 14);

CDR-H2 containing the amino acid sequence WIDPD X ₁ GN (SEQ ID NO: 135), where X ₁ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or Q;

CDR-H3 containing the amino acid sequence RLLMDY (SEQ ID NO: 16);

CDR-L1 containing the amino acid sequence stksl X ₂ hfngntyl (SEQ ID NO: 136), where X ₂ can be any of: an amino acid, an amino acid other than L, a conservative substitution L, L or S;

CDR-L2 containing the amino acid sequence yyms X ₃ (SEQ ID NO: 137), where X ₃ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or S; And

CDR-L3 containing the amino acid sequence qsleypft (SEQ ID NO: 19);

for example, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences of SEQ ID NO: 28, 29, 30, 31, 32 and 33, respectively, or SEQ ID NO: 28, 29, 30, 74, 75 and 76, respectively.

E51. An isolated antibody or antigen binding fragment thereof, from embodiment E50, wherein X ₁ is Q, X ₂ is S, and X ₃ is S.

E52. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence GFNI X ₁ DYYMN (SEQ ID NO: 175), where X ₁ can be any of: an amino acid, an amino acid other than K, a conservative substitution K, K or A;

CDR-H2 containing the amino acid sequence WIDPD X ₂ G X ₃ (SEQ ID NO: 176), where X ₂ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or Q; and X ₃ can be any of: an amino acid, an amino acid other than N, a conservative substitution for N, N or Q;

CDR-H3 containing the amino acid sequence RLLMDY (SEQ ID NO: 16);

CDR-L1 containing the amino acid sequence stks X ₄ X ₅ hfngn X ₆ yl (SEQ ID NO: 177), where X ₄ can be any of: an amino acid, an amino acid other than L, a conservative substitution, L or I; X ₅ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or S; and X ₆ can be any of: an amino acid, an amino acid other than T, a conservative substitution T, T or S;

CDR-L2 containing the amino acid sequence y X ₇ X ₈ s X ₉ (SEQ ID NO: 178), where X ₇ can be any of: an amino acid, an amino acid other than Y, a conservative substitution of Y, Y or A; X ₈ can be any of: an amino acid, an amino acid other than M, a conservative substitution of M, M or A; and X ₉ can be any of: an amino acid, an amino acid other than N, a conservative substitution for N, N, or S; And

CDR-L3 containing the amino acid sequence qs X ₁₀ X ₁₁ X ₁₂ p X ₁₃ t (SEQ ID NO: 197), where X ₁₀ can be any of: an amino acid, an amino acid other than L, a conservative substitution L, L or Y; X ₁₁ can be any of: an amino acid, an amino acid other than E, a conservative substitution of E, E or S; X ₁₂ can be any of: an amino acid, an amino acid other than Y, a conservative substitution of Y, Y or T; and X ₁₃ can be any of: an amino acid, an amino acid other than F, a conservative substitution F, F, L or W;

for example, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences of SEQ ID NO: 28, 29, 30, 31, 32 and 33, respectively, or SEQ ID NO: 28, 29, 30, 74, 75 and 76, respectively, optionally where:

X ₁ is Q, X ₂ is N, X ₃ is E, X ₄ is P, X ₅ is K, X ₆ is F and X ₇ is Q,

X ₈ is L, X ₉ is S and X ₁₀ is T,

X ₁₁ is Y, X ₁₂ is M, X ₁₃ is S and X ₁₄ is A, and/or

X ₁₅ is Q, X ₁₆ is L, X ₁₇ is E, X ₁₈ is Y and X ₁₉ is F.

E53. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence GFNIKDYYMN (SEQ ID NO: 14);

CDR-H2 containing the amino acid sequence WIDPD X ₁ GN (SEQ ID NO: 135), where X ₁ can be any of: an amino acid, an amino acid other than N, a conservative substitution N, N or Q;

CDR-H3 containing the amino acid sequence RLLMDY (SEQ ID NO: 16);

CDR-L1 containing the amino acid sequence stksl X ₂ hfngntyl (SEQ ID NO: 136), where X ₂ can be any of: an amino acid, an amino acid other than L, a conservative substitution L, L or S;

CDR-L2 containing the amino acid sequence yy X ₃ sX ₄ (SEQ ID NO: 179), where X ₃ can be any of: an amino acid, an amino acid other than M, a conservative substitution M, M or A; and X ₄ can be any of: an amino acid, an amino acid other than N, a conservative substitution for N, N or S; And

CDR-L3 containing the amino acid sequence qs X ₅ eypft (SEQ ID NO: 180), where X ₅ can be any of: an amino acid, an amino acid other than L, a conservative substitution of L, L or Y;

for example, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences of SEQ ID NO: 28, 29, 30, 31, 32 and 33, respectively, or SEQ ID NO: 28, 29, 30, 74, 75 and 76, respectively, optionally where:

X ₁ is Q,

X ₂ is S,

X ₃ is M and X ₄ is S, and/or

X ₅ is L.

E54. An isolated antibody or antigen binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from the VH region containing the amino acid sequence of SEQ ID NO: 6, wherein the CDR sequences are as defined by Chotia.

E55. An isolated antibody or antigen binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from the VL region containing the amino acid sequence of SEQ ID NO: 7, wherein the CDR sequences are as defined by Chotia.

E56. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from the VH region, comprising the amino acid sequence of SEQ ID NO: 6 and one, two or three CDR-L1 sequences from the VL region, containing the amino acid sequence of SEQ ID NO: 7, where the CDR sequences are as defined according to Chotia.

E57. An isolated antibody or antigen binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from a polypeptide encoded by nucleic acid sequence SEQ ID NO: 189, 190 or 191, wherein the CDR sequences are as defined by Chotia.

E58. An isolated antibody or antigen binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from a polypeptide encoded by the nucleic acid sequence SEQ ID NO: 185 or 186, wherein the CDR sequences are as defined by Chotia.

E59. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one, two or three CDR sequences from the polypeptide encoded by nucleic acid sequence SEQ ID NO: 189, 190 or 191, and one, two or three CDR sequences from a polypeptide encoded by the nucleic acid sequence SEQ ID NO: 185, 186, where the CDR sequences are as defined according to Chotia.

E60. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VH framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence having at least 80% , 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or, preferably, 100% sequence identity with the VH framework region of the VH region containing the amino acid sequence of any of SEQ ID NO: 6, 34-46, 88-91 or 93.

E61. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VL framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence having at least 80% , 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or, preferably, 100% sequence identity with the VL framework region of the VL region containing the amino acid sequence of any of SEQ ID NO: 7, 47-69 or 92.

E62. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VH framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence having at least 80% , 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity with the germline amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1 -69 or IGHV3-48.

E63. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VL framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence having at least 80% , 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to the germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3 -eleven.

E64. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VH framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence containing 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 substitutions relative to the VH framework region of the VH region containing the amino acid sequence, any of SEQ ID NO: 6, 34-46, 88-91 or 93.

E65. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VL framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence containing 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 substitutions relative to the VL framework region VL region containing the amino acid sequence, any of SEQ ID NO: 7, 47-69 or 92.

E66. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VH framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence containing 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 substitutions relative to the IGHV3-07 germline amino acid sequence, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69 or IGHV3-48.

E67. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises a VL framework region (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4) containing an amino acid sequence containing 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 substitutions relative to the IGKV1-39 germline amino acid sequence, IGKV2-28, IGKV2-30, IGKV4-1 or IGKV3-11.

E68. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, comprising a mouse IgG1 Fc region containing a substitution at one or more positions selected from E233, E318, K320 and R322 (e.g., E233P, E318A, K320A and R322A), for example wherein the mouse IgG1 Fc region contains one or more E233P, E318A, K320A and R322A substitutions numbered according to the Eu numbering scheme (see, for example, US Patent No. 5,624,821).

E69. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, comprising a human IgG1 Fc region containing substitutions at one or more positions selected from L234, L235, and G237 (e.g., L234A, L235A, and G237A), for example, wherein IgG1 Fc the human region contains one or more of the L234A, L235A and G237A substitutions, numbered according to the Eu numbering scheme.

E70. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, wherein the antibody further comprises a VH region comprising a germline variant VH amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3- 48, wherein the VH region contains one or more substitutions at positions T28, F29, A49, R72, N74, A75, and/or L79 (for example, one or more substitutions selected from T28N, F29I, A49G, R72A, N74T, A75S, and L79A), numbered according to the amino acid sequence of SEQ ID NO: 127, optionally, wherein the VH region contains the substitutions:

(i) T28N and F29I;

(ii) T28N, F29I and R72A;

(iii) T28N, F29I, R72A, A49G and L79A;

(iv) T28N, F29I, R72A, N74T and A75S; or

(v) T28N, F29I, R72A, A49G, L79A, N74T and A75S,

where (i)-(v) are as numbered according to the amino acid sequence of SEQ ID NO: 127, optionally where:

The VH region contains substitutions T28N, F29I, R72A, A49G, L79A, N74T and A75S, numbered according to the amino acid sequence SEQ ID NO: 127.

E71. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody further comprises a VH region comprising one or more (e.g., 2, 3, 4, 5, 6, or all) of the following:

(a) Asn at position 28,

(b) Ile at position 29,

(c) Gly at position 49,

(d) Ala at position 72,

(e) Thr at position 74,

(f) Ser at position 75, and

(g) Ala at position 79, numbered according to the amino acid sequence of SEQ ID NO: 127, optionally wherein the VH region contains:

(i) Asn at position 28 and Ile at position 29;

(ii) Asn at position 28, Ile at position 29, and Ala at position 72;

(iii) Asn at position 28, Ile at position 29, Ala at position 72, Gly at position 49, and Ala at position 79;

(iv) Asn at position 28, Ile at position 29, Ala at position 72, Thr at position 74, and Ser at position 75; or

(v) Asn at position 28, Ile at position 29, Ala at position 721, Gly at position 49, Ala at position 79, Thr at position 74 and Ser at position 75, numbered according to the amino acid sequence of SEQ ID NO: 127, optionally where :

The VH region contains Asn at position 28, Ile at position 29, Ala at position 72, Gly at position 49, Ala at position 79, Thr at position 743 and Ser at position 75, numbered according to the amino acid sequence of SEQ ID NO: 127.

E72. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody further comprises a VL region comprising a VL variant of the germline amino acid sequence IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1 or IGKV3-11, wherein VH region contains one or more substitutions at positions Y36 and/or L46 (e.g., Y36F and/or L46R), numbered according to the amino acid sequence of SEQ ID NO: 128, optionally wherein the VL region contains the substitutions:

(i) L46R; or

(ii) L46R and Y36F,

wherein (i)-(v) are numbered according to the amino acid sequence of SEQ ID NO: 128, optionally wherein the VL region contains the L46R substitution numbered according to the amino acid sequence of SEQ ID NO: 128.

E73. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody further comprises a VL region comprising one or more of the following:

(a) Tyr at position 36, and

(b) Leu at position 46, numbered according to the amino acid sequence of SEQ ID NO:128, optionally wherein the VL region contains:

(i) Leu at position 46; or

(ii) Leu at position 46 and Tyr at position 36, numbered according to the amino acid sequence of SEQ ID NO:128, optionally where:

The VL region contains Leu at position 46, numbered according to the amino acid sequence of SEQ ID NO:128.

E74. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising one or more CDRs from any of the above embodiments, wherein the one or more CDRs comprise at least one amino acid change, but no more than two, three, or four changes ( e.g. substitution, deletion or insertion, e.g. conservative substitution); where CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 do not contain the amino acid sequences SEQ ID NO: 22, 23, 24, 25, 26 and 27, respectively, SEQ ID NO: 22, 23, 24, 71, 72 and 73, respectively, SEQ ID NO: 28, 29, 30, 31, 32 and 33, respectively or SEQ ID NO: 28, 29, 30, 74, 75 and 76, respectively.

E75. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, further comprising a VH region containing the amino acid sequence shown in Table 1.

E76. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, further comprising a VL region containing the amino acid sequence shown in Table 1.

E77. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, comprising a VH region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NO: 6, 34-46, 88-91, or 93.

E78. An isolated antibody or antigen binding fragment thereof, from embodiment E77, comprising a VH region containing the amino acid sequence of SEQ ID NO: 6.

E79. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, comprising a VL region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NO: 7, 47-69, or 92.

E80. An isolated antibody or antigen binding fragment thereof, from embodiment E79, comprising a VL region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 100%) to the amino acid sequence of SEQ ID NO: 7.

E81. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a VH region containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NO: 6, 34-46, 88-91, or 93.

E82. An isolated antibody or antigen binding fragment thereof, from embodiment E81, comprising a VH region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 100%) to the amino acid sequence of SEQ ID NO: 6.

E83. An isolated antibody or antigen binding fragment thereof, from embodiment E81 or E82, further comprising a VL region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 100%) to the amino acid sequence of SEQ ID NO: 7.

E84. An isolated antibody or antigen binding fragment thereof, from embodiment E81, comprising a VH region containing the amino acid sequence of SEQ ID NO: 6.

E85. An isolated antibody or antigen binding fragment thereof, from embodiment E84, comprising a VL region containing the amino acid sequence of SEQ ID NO: 7.

E86. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a VL region containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NO: 7, 47-69, or 92.

E87. An isolated antibody or antigen binding fragment thereof, from embodiment E86, comprising a VL region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 100%) to the amino acid sequence of SEQ ID NO: 7.

E88. An isolated antibody or antigen binding fragment thereof, from embodiment E86 or E87, further comprising a VH region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% sequence identity (e.g., 100%) to the amino acid sequence of SEQ ID NO: 6.

E89. An isolated antibody or antigen binding fragment thereof, from embodiment E86, comprising a VL region comprising an amino acid sequence selected from SEQ ID NO: 7, 47-69, or 92.

E90. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a VH region containing the amino acid sequence of SEQ ID NO: 39, wherein one or more amino acid residues of said SEQ ID NO: 39 contains one or more amino acid substitutions selected from K30A , N55Q, N57Q, D61E, P62A, K63A and F64V, numbered according to SEQ ID NO: 39.

E91. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a VH region comprising at least one of the following:

(a) Ala at position 30

(b) Gln at position 55,

(c) Gln at position 57,

(d) Glu at position 61,

(e) Ala at position 62,

(f) Ala at position 63, and

(g) Val at position 64, numbered according to SEQ ID NO: 39.

E92. An isolated antibody or antigen binding fragment thereof, from embodiment E90, wherein SEQ ID NO: 39 comprises:

(i) N55Q and D61E; or

(ii) N55Q, D61E and F64V, numbered as per SEQ ID NO: 39, optionally wherein said SEQ ID NO: 39 contains substitutions N55Q and D61E, numbered as per SEQ ID NO: 39.

E93. An isolated antibody or antigen binding fragment thereof, from embodiment E91, wherein the VH region comprises:

(i) Gln at position 55 and Glu at position 61; or

(ii) Gln at position 55, Glu at position 61 and Val at position 64, numbered according to SEQ ID NO: 39, optionally wherein the VH region contains Gln at position 55 and Glu at position 61, numbered according to SEQ ID NO: 39.

E94. An isolated antibody or antigen binding fragment thereof, from embodiment E90 or E92, further comprising a VL region comprising the amino acid sequence of SEQ ID NO: 47, wherein one or more amino acid residues of said SEQ ID NO: 47 contains one or more amino acid substitutions selected from L30S , Y55A, M56A, N58S, A60Q, M94Q, L97Y, F101L, F101W and Q105G, or any combination thereof, numbered according to SEQ ID NO: 47, optionally, wherein one or more amino acid residues of said SEQ ID NO: 47 contains one or more amino acid residues substitutions selected from L30S, M56A, N58S, M94Q, L97Y and Q105G.

E95. An isolated antibody or antigen binding fragment thereof, from embodiment E91 or E93, further comprising a VL region comprising at least one of the following:

(a) Ser at position 30,

(b) Ala at position 55,

(c) Ala at position 56,

(d) Ser at position 58,

(e) Gln at position 60,

(f) Gln at position 94,

(g) Tyr at position 97,

(h) Leu at position 101,

(i) Trp at position 101 and

(j) Gly at position 105, numbered according to SEQ ID NO: 47, optionally wherein the VL region contains at least one of the following:

(a) Ser at position 30,

(b) Ala at position 56,

(c) Ser at position 58,

(d) Gln at position 94,

(e) Tyr at position 97 and

(f) Gly at position 105.

E96. An isolated antibody or antigen binding fragment thereof, from embodiment E94, wherein SEQ ID NO: 47 contains the L30S, M56A, N58S, M94Q, L97Y and/or Q105G substitution numbered according to SEQ ID NO: 47.

E97. An isolated antibody or antigen binding fragment thereof, from embodiment E95, wherein the VL region contains Ser at position 30, Ala at position 56, Ser at position 58, Gln at position 94, Tyr at position 97, and/or Gly at position 105, numbered according to SEQ ID NO: 47.

E98. An isolated antibody or antigen-binding fragment thereof, from embodiment E94, wherein SEQ ID NO: 47 contains the L30S, N58S, M94Q and/or Q105G substitution numbered according to SEQ ID NO: 47, optionally wherein SEQ ID NO: 47 contains all of L30S, N58S, M94Q and Q105G replace.

E99. An isolated antibody or antigen binding fragment thereof, from embodiment E95, wherein the VL region contains Ser at position 30, Ser at position 58, Gln at position 94, and/or Gly at position 105, numbered according to SEQ ID NO: 47, optionally wherein the VL region contains all of: Ser at position 30, Ser at position 58, Gln at position 94, and Gly at position 105.

E100. An isolated antibody or antigen binding fragment thereof, from embodiment E94, wherein SEQ ID NO: 39 contains the N55Q and D61E substitutions numbered according to SEQ ID NO: 39 and where SEQ ID NO: 47 contains the L30S, N58S, M94Q and Q105G substitutions numbered according to SEQ ID NO: 47.

E101. An isolated antibody or antigen binding fragment thereof, from embodiment E95, wherein the VH region contains Gln at position 55 and Glu at position 61, numbered according to SEQ ID NO: 39 and the VL region contains Ser at position 30, Ser at position 58, Gln at position 94 and Gly at position 105, numbered according to SEQ ID NO: 47.

E102. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a VL region containing the amino acid sequence of SEQ ID NO: 47, wherein one or more amino acid residues of said SEQ ID NO: 47 contains one or more amino acid substitutions selected from L30S . one or more amino acid residues of the specified SEQ ID NO: 47 contains one or more amino acid substitutions selected from L30S, M56A, N58S, M94Q, L97Y and Q105G.

103. An isolated antibody or antigen binding fragment thereof, from embodiment E102, wherein SEQ ID NO: 47 contains the aL30S, N58S, M94Q and/or Q105G substitution numbered according to SEQ ID NO: 47.

E104. An isolated antibody or antigen binding fragment thereof, from embodiment E102, further comprising a VH region containing the amino acid sequence of SEQ ID NO: 39, wherein the sequence of SEQ ID NO: 39 contains one or more amino acid substitutions selected from K30A, N55Q, N57Q, D61E, P62A, K63A and F64V or any combination of those numbered as per SEQ ID NO: 39.

E105. An isolated antibody or antigen binding fragment thereof, from embodiment E104, wherein SEQ ID NO: 39 contains the N55Q and D61E substitutions.

E106. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, comprising a heavy chain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 or 3.

E107. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, further comprising a light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5.

E108. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 6 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 7.

E109. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 2 or 3, and a light chain containing the amino acid sequence of SEQ ID NO: 5.

E110. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing

(a) a light chain containing the amino acid sequence of SEQ ID NO: 5, and

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 3, with or without a C-terminal lysine residue.

E111. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a heavy chain containing the amino acid sequence encoded by the plasmid insert stored in ATCC and having accession number PTA-124917, a light chain containing the amino acid sequence encoded by the plasmid insert stored in ATCC and having accession number PTA-124918, or both.

E112. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a heavy chain containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% (e.g., 100%) sequence identity to SEQ ID NO: 2 or 3, optionally, where the heavy chain contains the amino acid sequence of SEQ ID NO: 2.

E113. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a light chain containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% (e.g., 100%) sequence identity to SEQ ID NO: 5, optionally, wherein the light chain contains the amino acid sequence of SEQ ID NO: 5, optionally wherein the isolated antibody or thereof the antigen-binding fragment further comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 2.

E114. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a heavy chain comprising the amino acid sequence comprising SEQ ID NO: 2 and a light chain containing the amino acid sequence comprising SEQ ID NO: 5.

E115. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising a heavy chain comprising the amino acid sequence comprising SEQ ID NO: 3 and a light chain containing the amino acid sequence comprising SEQ ID NO: 5.

E116. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody further comprises a VH framework region (e.g., one, two, three or all four of FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence identity to the VH framework region of the VH region containing the amino acid sequence of any of SEQ ID NO: 6, 34-46, 88-91 or 93, or amino acid sequence having at least one but no more than twenty changes, amino acid substitutions or deletions, in the amino acid sequence of the entire VH framework region (including FR1, FR2, FR3 and FR4).

E117. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody further comprises a VL framework region (e.g. FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity to the VL framework region of the VL region containing the amino acid sequence of any of SEQ ID NO: 7, 47-69 or 92, or an amino acid sequence having at least one, two, three, four, five, six, seven, ten, fifteen, but less than twenty changes, for example, amino acid substitutions or deletions of the amino acid sequence of the entire VL framework region (including FR1, FR2, FR3 and FR4).

E118. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody comprises a VH framework region (e.g. FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98 % or 100% sequence identity to the VH framework region in the germline amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69 or IGHV3-48, or an amino acid sequence having at least one, two , three, four, five, six, seven, ten, fifteen, but less than twenty changes, for example, amino acid substitutions or deletions of the amino acid sequence of the entire VH framework region (including FR1, FR2, FR3 and FR4).

E119. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

where the antibody contains a VH region containing a VH variant of the germline amino acid sequence IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69 or IGHV3-48, where the VH region contains one or more substitutions at positions T28, F29 , A49, R71, N73, A74 and/or L78 (e.g., one or more substitutions selected from T28N, F29I, A49G, R72A, N74T, A75S and L79A), numbered according to the amino acid sequence of SEQ ID NO: 127, optionally where VH area contains replacements:

(i) T28N and F29I;

(ii) T28N, F29I and R72A;

(iii) T28N, F29I, R72A, A49G and L79A;

(iv) T28N, F29I, R72A, N74T and A75S; or

(v) T28N, F29I, R72A, A49G, L79A, N74T and A75S,

where (i)-(v) are numbered according to the amino acid sequence of SEQ ID NO: 127, optionally where:

The VH region contains substitutions T28N, F29I, R72A, A49G, L79A, N74T and A75S, numbered according to the amino acid sequence SEQ ID NO: 127.

E120. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody contains a VH region containing one or more (e.g., 2, 3, 4, 5, 6, or all) of the following:

(a) Asn at position 28,

(b) Ile at position 29,

(c) Gly at position 49,

(d) Ala at position 72,

(e) Thr at position 74,

(f) Ser at position 75, and

(g) Ala at position 79, numbered according to the amino acid sequence of SEQ ID NO: 127, optionally wherein the VH region contains:

(i) Asn at position 28 and Ile at position 29;

(ii) Asn at position 28, Ile at position 29, and Ala at position 72;

(iii) Asn at position 28, Ile at position 29, Ala at position 72, Gly at position 49, and Ala at position 79;

(iv) Asn at position 28, Ile at position 29, Ala at position 72, Thr at position 74, and Ser at position 75; or

(v) Asn at position 28, Ile at position 29, Ala at position 72, Gly at position 49, Ala at position 79, Thr at position 74 and Ser at position 75, numbered according to the amino acid sequence of SEQ ID NO: 127, optionally wherein :

The VH region contains Asn at position 28, Ile at position 29, Ala at position 72, Gly at position 49, Ala at position 79, Thr at position 74 and Ser at position 75, numbered according to the amino acid sequence of SEQ ID NO: 127.

E121. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody comprises a VL framework region (e.g. FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98 % or 100% sequence identity to the germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1 or IGKV3-11, or an amino acid sequence having at least one, two, three, four, five, six , seven, ten, fifteen, but less than twenty changes, for example, amino acid substitutions or deletions of the amino acid sequence of the entire VL framework region (including FR1, FR2, FR3 and FR4).

E122. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody contains a VL region containing a VL variant of the germline amino acid sequence IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1 or IGKV3-11, wherein the VL region contains one or more substitutions at positions Y36 and/or L46 (e.g. , Y36F and/or L46R), numbered according to the amino acid sequence of SEQ ID NO: 128, optionally wherein the VL region contains the substitutions:

(i) L46R; or

(ii) L46R and Y36F,

wherein (i) and (ii) are numbered according to the amino acid sequence of SEQ ID NO: 128, optionally wherein the VL region contains the L46R substitution numbered according to the amino acid sequence of SEQ ID NO: 128.

E123. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

wherein the antibody contains a VL region containing one or both of the following:

(a) Tyr at position 36, and

(b) Leu at position 46, numbered according to the amino acid sequence of SEQ ID NO: 128, optionally wherein the VL region contains:

(i) Leu at position 46; or

(ii) Leu at position 46 and Tyr at position 36, numbered according to the amino acid sequence of SEQ ID NO: 128, optionally where:

The VL region contains Leu at position 46, numbered according to the amino acid sequence SEQ ID NO: 128.

E124. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

further comprising a mouse IgG1 Fc region containing one or more substitutions selected from positions E233, E318, K320, and R322 (e.g., E233P, E318A, K320A, and R322A) numbered according to the Eu numbering scheme, e.g., relative to the mouse IgG1 Fc shown in the table 1.

E125. An isolated antibody or antigen binding fragment thereof, from embodiment E124, wherein the mouse IgG1 Fc region contains E233P, E318A, K320A and R322A substitutions numbered according to the Eu numbering scheme, for example, relative to the mouse IgG1 Fc shown in Table 1.

E126. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 25,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 27; And

further comprising a human IgG1 Fc region containing one or more substitutions selected from positions L234, L235, and G237 (e.g., L234A, L235A, and G237A), numbered according to the Eu numbering scheme, for example, relative to the human IgG1 Fc shown in Table 1.

E127. An isolated antibody or antigen binding fragment thereof, from embodiment E126, wherein the human IgG1 Fc region contains L234A, L235A and G237A substitutions numbered according to the Eu numbering scheme, for example, relative to the human IgG1 Fc shown in Table 1.

E128. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 71,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 72, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 73; And

wherein the antibody further comprises a VH framework region (e.g. one, two, three or four or FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93 %, 95%, 97%, 98%, or preferably, 100% sequence identity with the VH framework region of the VH region containing the amino acid sequence of any of SEQ ID NO: 6, 34-46, 88-91 or 93.

E129. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 71,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 72, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 73; And

wherein the antibody further comprises a VL framework region (e.g., one, two, three or four or FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93 %, 95%, 97%, 98%, or preferably, 100% sequence identity to the VL framework region of the VL region containing the amino acid sequence of any of SEQ ID NO: 7, 47-69 or 92.

E130. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 71,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 72, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 73; And

wherein the antibody further comprises a VH framework region (e.g. one, two, three or four or FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93 %, 95%, 97%, 98%, or 100% sequence identity to the VH framework region in the germline amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48.

E131. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 71,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 72, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 73; And

wherein the antibody further comprises a VL framework region (e.g. one, two, three or four or FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93 %, 95%, 97%, 98%, or 100% sequence identity to the germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11.

E132. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 71,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 72, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 73; And

further comprising a mouse IgG1 Fc region containing one or more substitutions selected from positions E233, E318, K320, and R322 (e.g., E233P, E318A, K320A, and R322A) numbered according to the Eu numbering scheme, for example, relative to the mouse IgG1 Fc shown in Table 1.

E133. An isolated antibody or antigen binding fragment thereof, from embodiment E132, wherein the mouse IgG1 Fc region contains E233P, E318A, K320A and R322A substitutions numbered according to the Eu numbering scheme, for example, relative to the mouse IgG1 Fc shown in Table 1.

E134. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 22,

CDR-H2 containing the amino acid sequence SEQ ID NO: 23,

CDR-H3 containing the amino acid sequence SEQ ID NO: 24,

CDR-L1 containing the amino acid sequence SEQ ID NO: 71,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 72, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 73; And

additionally containing a human IgG1 Fc region containing L234A, L235A and G237A substitutions numbered according to the Eu numbering scheme, for example, relative to the human IgG1 Fc presented in Table 1.

E135. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 28,

CDR-H2 containing the amino acid sequence SEQ ID NO: 29,

CDR-H3 containing the amino acid sequence SEQ ID NO: 30,

CDR-L1 containing the amino acid sequence SEQ ID NO: 31,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 32, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 33; And

wherein the antibody further comprises a VH framework region (e.g., one, two, three or four of FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence identity to the VH framework region of the VH region containing the amino acid sequence of any of SEQ ID NO: 6, 34-46, 88-91 or 93.

E136. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 28,

CDR-H2 containing the amino acid sequence SEQ ID NO: 29,

CDR-H3 containing the amino acid sequence SEQ ID NO: 30,

CDR-L1 containing the amino acid sequence SEQ ID NO: 74,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 75, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 76; And

wherein the antibody further comprises a VH framework region (e.g., one, two, three or four of FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93 %, 95%, 97%, 98%, or preferably, 100% sequence identity with the VH framework region of the VH region containing the amino acid sequence of any of SEQ ID NO: 6, 34-46, 88-91 or 93.

E137. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 28,

CDR-H2 containing the amino acid sequence SEQ ID NO: 29,

CDR-H3 containing the amino acid sequence SEQ ID NO: 30,

CDR-L1 containing the amino acid sequence SEQ ID NO: 31,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 32, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 33; And

wherein the antibody further comprises a VL framework region (e.g., one, two, three or four of FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence identity to the VL framework region of the VL region containing the amino acid sequence of any of SEQ ID NO: 7, 47-69 or 92.

E138. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 28,

CDR-H2 containing the amino acid sequence SEQ ID NO: 29,

CDR-H3 containing the amino acid sequence SEQ ID NO: 30,

CDR-L1 containing the amino acid sequence SEQ ID NO: 31,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 32, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 33; And

wherein the antibody further comprises a VH framework region (e.g., one, two, three or four of FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to the germline amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48.

E139. An isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 28,

CDR-H2 containing the amino acid sequence SEQ ID NO: 29,

CDR-H3 containing the amino acid sequence SEQ ID NO: 30,

CDR-L1 containing the amino acid sequence SEQ ID NO: 31,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 32, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 33; And

wherein the antibody further comprises a VL framework region (e.g., one, two, three or four of FR1, FR2, FR3 or FR4) containing an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to the germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11.

E140. An isolated antibody or antigen-binding fragment thereof, containing:

CDR-H1 containing the amino acid sequence SEQ ID NO: 28,

CDR-H2 containing the amino acid sequence SEQ ID NO: 29,

CDR-H3 containing the amino acid sequence SEQ ID NO: 30,

CDR-L1 containing the amino acid sequence SEQ ID NO: 31,

CDR-L2 containing the amino acid sequence of SEQ ID NO: 32, and

CDR-L3 containing the amino acid sequence SEQ ID NO: 33; And

further comprising a human IgG1 Fc region containing one or more substitutions selected from positions L234, L235 and G237 (e.g., L234A, L235A and G237A), numbered according to the Eu numbering scheme, for example, relative to the human IgG1 Fc shown in Table 1.

E141. An isolated antibody or antigen binding fragment thereof, from embodiment E140, wherein the human IgG1 Fc region contains L234A, L235A and G237A substitutions numbered according to the Eu numbering scheme, for example, relative to the human IgG1 Fc shown in Table 1.

E142. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody is a multispecific antibody (eg, a bispecific antibody).

E143. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody is a multivalent antibody (eg, a divalent antibody).

E144. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody is a humanized antibody, a human antibody, a mouse antibody, a chimeric antibody, or a camel antibody.

E145. An isolated antibody or antigen-binding fragment thereof that specifically binds to αvβ8 integrin, comprising a VH region and a VL region, wherein the VH region and VL region contain the amino acid sequences:

(i) SEQ ID NO: 6 and 7, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(ii) SEQ ID NO: 34 and 65, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(iii) SEQ ID NO: 34 and 62, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(iv) SEQ ID NO: 34 and 66, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(v) SEQ ID NO: 34 and 63, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(vi) SEQ ID NO: 34 and 64, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(vii) SEQ ID NO: 37 and 65, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(viii) SEQ ID NO: 37 and 62, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(ix) SEQ ID NO: 37 and 66, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(x) SEQ ID NO: 37 and 63, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xi) SEQ ID NO: 37 and 64, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xii) SEQ ID NO: 36 and 65, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xiii) SEQ ID NO: 36 and 62, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xiv) SEQ ID NO: 36 and 66, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xv) SEQ ID NO: 36 and 63, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xvi) SEQ ID NO: 36 and 64, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xvii) SEQ ID NO: 35 and 65, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xviii) SEQ ID NO: 35 and 62, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xix) SEQ ID NO: 35 and 66, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xx) SEQ ID NO: 35 and 63, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxi) SEQ ID NO: 35 and 64, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxii) SEQ ID NO: 38 and 65, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxiii) SEQ ID NO: 38 and 62, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxiv) SEQ ID NO: 38 and 66, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxv) SEQ ID NO: 38 and 63, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxvi) SEQ ID NO: 38 and 64, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxvii) SEQ ID NO: 20 and 21, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxviii) SEQ ID NO: 88 and 47, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxix) SEQ ID NO: 89 and 47, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxx) SEQ ID NO: 90 and 47, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxi) SEQ ID NO: 90 and 92, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxii) SEQ ID NO: 39 and 47, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxiii) SEQ ID NO: 6 and 67, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxiv) SEQ ID NO: 6 and 68, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxv) SEQ ID NO: 6 and 69, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxvi) SEQ ID NO: 93 and 67, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxvii) SEQ ID NO: 93 and 68, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them;

(xxxviii) SEQ ID NO: 93 and 69, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them; or

(xxxix) SEQ ID NO: 93 and 7, respectively, or an amino acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or preferably 100% sequence identity with them, optimally, where:

VH region and VL region contain amino acid sequences

(i) SEQ ID NO: 6 and 7, respectively;

(ii) SEQ ID NO: 34 and 65, respectively;

(iii) SEQ ID NO: 34 and 62, respectively;

(iv) SEQ ID NO: 34 and 66, respectively;

(v) SEQ ID NO: 34 and 63, respectively;

(vi) SEQ ID NO: 34 and 64, respectively;

(vii) SEQ ID NO: 37 and 65, respectively;

(viii) SEQ ID NO: 37 and 62, respectively;

(ix) SEQ ID NO: 37 and 66, respectively;

(x) SEQ ID NO: 37 and 63, respectively;

(xi) SEQ ID NO: 37 and 64, respectively;

(xii) SEQ ID NO: 36 and 65, respectively;

(xiii) SEQ ID NO: 36 and 62, respectively;

(xiv) SEQ ID NO: 36 and 66, respectively;

(xv) SEQ ID NO: 36 and 63, respectively;

(xvi) SEQ ID NO: 36 and 64, respectively;

(xvii) SEQ ID NO: 35 and 65, respectively;

(xviii) SEQ ID NO: 35 and 62, respectively;

(xix) SEQ ID NO: 35 and 66, respectively;

(xx) SEQ ID NO: 35 and 63, respectively;

(xxi) SEQ ID NO: 35 and 64, respectively;

(xxii) SEQ ID NO: 38 and 65, respectively;

(xxiii) SEQ ID NO: 38 and 62, respectively;

(xxiv) SEQ ID NO: 38 and 66, respectively;

(xxv) SEQ ID NO: 38 and 63, respectively;

(xxvi) SEQ ID NO: 38 and 64, respectively;

(xxvii) SEQ ID NO: 20 and 21, respectively;

(xxviii) SEQ ID NO: 88 and 47, respectively;

(xxix) SEQ ID NO: 89 and 47, respectively;

(xxx) SEQ ID NO: 90 and 47, respectively;

(xxxi) SEQ ID NO: 90 and 92, respectively;

(xxxii) SEQ ID NO: 39 and 47, respectively;

(xxxiii) SEQ ID NO: 6 and 67, respectively;

(xxxiv) SEQ ID NO: 6 and 68, respectively;

(xxxv) SEQ ID NO: 6 and 69, respectively;

(xxxvi) SEQ ID NO: 93 and 67, respectively;

(xxxvii) SEQ ID NO: 93 and 68, respectively;

(xxxviii) SEQ ID NO: 93 and 69, respectively; or

(xxxix) SEQ ID NO: 93 and 7, respectively.

E146. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which contains or has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE.

E147. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, wherein the antibody belongs to an isotype selected from IgG1, IgG2, IgG3, IgG4, or any variant thereof.

E148. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, wherein the antibody comprises an IgG1 or IgG2 heavy chain constant region (eg, human IgG1 or human IgG2).

E149. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which contains or has a human IgG1 heavy chain constant region.

E150. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which contains or has a light chain constant region selected from, for example, kappa or lambda light chain constant regions.

E151. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which contains or has a kappa (eg, human kappa) light chain constant region.

E152. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which comprises a heavy chain Fc region having an altered hinge region to reduce effector cell function.

E153. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, which has reduced antibody-dependent cellular cytotoxicity (ADCC) and/or reduced complement-dependent cytotoxicity (CDC).

E154. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which contains a hinge region having a substitution at at least one position of L234, L235 or G237, for example, compared to human IgG1, numbered according to the Eu numbering scheme.

E155. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, comprising a human IgG1 Fc region containing at least one substitution selected from L234A, L235A and G237A, numbered according to the Eu numbering scheme.

E156. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, which has a hinge region comprising the amino acid sequence EPKSCDKTHTCPPCPAPEAAGAP (SEQ ID NO: 126).

E157. An isolated antibody or antigen-binding fragment thereof, from any of the above embodiments, that is modified to remove an immunogenic T-cell epitope.

E158. An isolated antibody or antigen binding fragment thereof, from embodiment E157, which comprises a VL containing at least one substitution selected from the group consisting of L30S, N58S, M56A, M94Q, L97Y and Q105G, numbered according to SEQ ID NO: 47.

E159. An antibody or antigen binding fragment thereof, from any of the above embodiments, wherein said antibody or antigen binding fragment thereof has at least one of the following properties:

(i) the binding affinity, expressed as KD, for human αvβ8 integrin is less than that for mouse ADWA11 antibody, eg, less than 536 pM;

(ii) a K _D for human αvβ8 integrin that is less than or equal to 100 pM for purified human αvβ8 integrin;

(iii) K _D for mouse αvβ8 integrin, which is less than 100 pM;

(iv) K _D for cynomolgus αvβ8 integrin, which is less than 100 pM;

(v) K _D for rat αvβ8 integrin, which is approximately 160 pM;

(vi) approximately equivalent affinity for at least two, three or all human, cynomolgus, mouse and rat αvβ8 integrins, for example, with a K _D of less than 100 pM, for example, determined using the Biacore affinity assay;

(vii) an IC ₅₀ for inhibition of TGFβ transactivation that is less than 183 pM;

(viii) IC ₅₀ for inhibition of TGFβ transactivation in U251 cells, which is from about 100 pM to about 300 pM;

(ix) EC ₅₀ for U251 cells from about 100 pM to about 400 pM pM;

(x) EC ₅₀ for C8-S cells from about 110 pM to about 180 pM;

(xi) at least one predicted human pharmacokinetic (PK) parameter selected from:

A. clearance from the central chamber (CL) is approximately 0.12-0.15 ml/h/kg;

b. intercompartmental clearance distribution (CLF) approximately 0.15-0.51 ml/h/kg;

With. volume of distribution for the central chamber (V1) approximately 36-39 ml/kg;

d. volume of distribution for the peripheral chamber (V2) approximately 21-33 ml/kg; and/or

e. the final half-life (t _1/2 ) is approximately 12-17 days; And

(xii) lack of detectable binding to human Fcγ or C1q receptors.

E160. An antibody or antigen binding fragment thereof, from any of the above embodiments, wherein said antibody or antigen binding fragment thereof has at least one of the following properties:

(i) binds specifically to αvβ8 integrin, but not to other integrins;

(ii) reduces the interaction between αvβ8 integrin and latency-associated peptide (LAP);

(iii) reduces TGF-β signaling;

(iv) effectively blocks αvβ8 integrin-mediated TGFβ activation with an IC ₅₀ ≤10 nM;

(v) has a comparable Kd (within 5-fold) to the non-human primate ortholog (NHP);

(vi) selectively binds human αvβ8 and does not detectably bind αvβ8 homolog (eg, αvβ1, αvβ3, αvβ5, and αvβ6);

(vii) causes suppression of growth and/or complete regression of a tumor in a human or in an animal model of cancer, for example, squamous cell carcinoma, breast cancer and/or colon cancer, alone or in combination with an immunomodulatory agent, for example, a checkpoint inhibitor modulator, e.g. , inhibitors of PD-1, PD-L1, CTLA-4 or a stimulatory molecule agonist, for example, 4-1BB;

(viii) causes suppression of growth and/or complete tumor regression in an animal model of cancer in combination with anticancer therapy, such as radiotherapy;

(ix) shows at least a 60% reduction in tumor growth in a syngeneic tumor xenograft model, e.g., when administered at a dose of ≤10 mg/kg, alone or in combination with an immunomodulatory agent (e.g., a PD-1, PD-L1, or CTLA inhibitor -4);

(x) enhances an antitumor response in the presence of one or more immunomodulators, e.g., a checkpoint inhibitor antagonist, e.g., a PD-1, PD-L1, or CTLA-4 antagonist, or an immune response activator, e.g., a 4-1BB agonist, when administered to a subject , for example, a mouse or a person;

(xi) has an efficacy that is independent of αvβ8 integrin expression in a tumor model;

(xii) can increase the abundance of CD8+ GzmB+ T cells in the tumor microenvironment, for example, as monotherapy;

(xiii) shows a reduction, e.g., at least >80% reduction in tumor growth when used in combination with a checkpoint inhibitor antagonist (e.g., anti-PD-1 or anti-PD-L1 antibody), e.g., in a syngeneic model of squamous cell carcinoma, breast cancer and/or colon cancer;

(xiv) demonstrates a statistically significant improvement in overall subject survival by Kaplan-Meier analysis;

(xv) demonstrates a high degree of thermal stability;

(xvi) shows minimal aggregation at high concentrations; And

(xvii) can demonstrate reproducible expression and purity under industrial production conditions.

E161. An antibody or antigen binding fragment thereof, from any of the above embodiments, wherein said antibody or antigen binding fragment thereof has at least one of the following properties:

(i) a binding affinity, expressed as _KD , for human αvβ8 integrin that is less than the _KD for mouse ADWA11 antibody, eg, less than 536 pM;

(ii) a K _D for human αvβ8 integrin that is less than or equal to 100 pM for purified human αvβ8 integrin;

(iii) K _D for mouse αvβ8 integrin, which is less than 100 pM;

(iv) K _D for cynomolgus αvβ8 integrin, which is less than 100 pM;

(v) K _D for rat αvβ8 integrin, which is approximately 160 pM;

(vi) approximately equivalent affinity for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, for example, with a K _D that is less than 100 pM determined using the Biacore affinity assay;

(vii) an IC ₅₀ for inhibition of TGFβ transactivation that is less than 183 pM;

(viii) IC ₅₀ for inhibition of TGFβ transactivation in U251 cells from about 100 pM to about 300 pM;

(ix) EC ₅₀ for U251 cells is approximately 126 pM with a standard deviation of plus/minus 34 pM;

(x) EC ₅₀ for U251 cells is approximately 256 pM with a standard deviation of plus/minus 115 pM;

(xi) EC ₅₀ for U251 cells from about 80 pM to about 400 pM;

(xii) EC ₅₀ for C8-S cells is approximately 115 pM;

(xiii) EC ₅₀ for C8-S cells is approximately 145 pM with a standard deviation of plus/minus 23.7 pM;

(xiv) EC ₅₀ for C8-S cells from about 110 pM to about 180 pM;

(xv) at least one predicted human pharmacokinetic (PK) parameter selected from:

A. clearance from the central chamber (CL) is approximately 0.12-0.15 ml/h/kg;

b. intercompartmental clearance distribution (CLF) approximately 0.15-0.51 ml/h/kg;

With. volume of distribution for the central chamber (V1) approximately 36-39 ml/kg;

d. volume of distribution for the peripheral chamber (V2) is approximately 21-33 1 ml/kg; and/or

e. the final half-life (t _1/2 ) is approximately 12-17 days; And

(xvi) lack of detectable binding to human Fcγ or C1q receptors.

E162. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, that binds human αvβ8 integrin with a K _D of less than or equal to 100 pM for purified human αvβ8 integrin.

E163. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, that binds human αvβ8 integrin with a K _D of less than 536 pM.

E164. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, that inhibits TGFβ activation with an IC ₅₀ of less than 183 pM.

E165. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, that inhibits TGFβ activation with an IC50 of 100 pM to about 300 pM.

E166. An isolated antibody or antigen binding fragment thereof, from any of the above embodiments, that inhibits TGFβ activation in U251 cells with an IC50 of 199 +/- 93.6 pM.

E167. A pharmaceutical composition comprising an antibody or antigen binding fragment thereof from any of the above embodiments, and a pharmaceutically acceptable carrier or excipient.

E168. A nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof, as in any one of embodiments E1-E166.

E169. A nucleic acid molecule containing:

(i) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO: 1, 183, 189 or 191 and the coding heavy chain;

(ii) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO:190 and a coding variable region heavy chain;

(iii) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO: 192 or 193 and coding heavy chain constant region; or

(iv) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity with the nucleic acid sequence of the plasmid insert deposited in the ATCC and having access number PTA-124917.

E170. A nucleic acid molecule containing:

(i) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO: 4 or 185 and coding light chain;

(ii) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO:186 and a coding variable region light chain;

(iii) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO: 194 and a coding constant region light chain; or

(iv) a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity with the nucleic acid sequence of the plasmid insert deposited in the ATCC and having access number PTA-124918.

E171. A nucleic acid molecule containing a nucleotide sequence having at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 1 or 183 , and a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 4.

E172. A nucleic acid molecule containing a nucleotide sequence having at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 189 or 191 , and a nucleotide sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 185.

E173. A vector containing a nucleic acid molecule according to any one of embodiments E168-E172.

E174. A host cell comprising the nucleic acid molecule of any one of embodiments E168-E172 or the vector of embodiment E173.

E175. The host cell of embodiment E174, wherein the host cell is a mammalian cell, such as a human cell.

E176. The host cell of embodiment E175, wherein the host cell is a CHO cell, a COS cell, a HEK-293 cell, an NS0 cell, a PER.C6® cell, or a Sp2.0 cell.

E177. A method of producing an isolated antibody or antigen-binding fragment thereof that specifically binds to human αvβ8 integrin, comprising culturing a host cell according to any one of embodiments E174-E176, under conditions wherein said antibody or antigen-binding fragment thereof is expressed by said host cell.

E178. The method of embodiment E177, further comprising isolating the antibody or antigen binding fragment thereof.

E179. A method of reducing TGFβ signaling in a subject in need thereof, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof of any one of embodiments E1-E166 or a pharmaceutical composition of any of embodiments E167.

E180. A method of reducing αvβ8 integrin activity in a subject in need thereof, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof of any one of embodiments E1-E166 or a pharmaceutical composition of any of embodiments E167.

E181. A method of treating a disease, disorder, or condition associated with or mediated by abnormal (e.g., elevated) TGFβ signaling, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any one of embodiments E1-E166 or a pharmaceutical composition of any of embodiments E167.

E182. A method of inducing an antitumor response in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any one of embodiments E1-E166 or a pharmaceutical composition of any of embodiments E167, optionally wherein the antibody or antigen-binding fragment thereof is administered in combination with a second therapy, optionally wherein the antibody or antigen binding fragment thereof and the second therapy are administered simultaneously, sequentially or separately, optionally wherein:

(i) the antibody or antigen-binding fragment thereof is administered prior to administration of the second therapy, or

(ii) the antibody or antigen-binding fragment thereof is administered after the administration of the second therapy.

E183. The antibody or antigen binding fragment thereof of any one of embodiments E1-E166 or the pharmaceutical composition of embodiment E167 for use in reducing αvβ8 integrin activity in a subject.

E184. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof of any one of embodiments E1-E166 or a pharmaceutical composition of any of embodiments E167.

E185. The method of embodiment E184, wherein said antibody or antigen binding fragment thereof has at least one of the following properties:

(i) binds specifically to αvβ8 integrin (eg, human, mouse, cynomolgus and/or rat αvβ8 integrin);

(ii) reduces the interaction between αvβ8 integrin and latency-associated peptide (LAP);

(iii) reduces TGF-β signaling;

(iv) effectively blocks αvβ8 integrin-mediated TGFβ activation with an IC50 ≤10 nM;

(v) has a comparable Kd (within 5-fold) to the non-human primate ortholog (NHP);

(vi) selectively binds human αvβ8 and does not detectably bind αvβ8 homolog (eg, αvβ1, αvβ3, αvβ5, and αvβ6);

(vii) causes suppression of growth and/or complete regression of a tumor in an animal model of cancer selected from, for example, squamous cell carcinoma, breast cancer and colon cancer, alone or in combination with an immunomodulatory agent, for example, a modulator of checkpoint inhibitors, for example, inhibitors PD-1, PD-L1, CTLA-4 or a stimulatory molecule agonist such as 4-1BB;

(viii) causes suppression of growth and/or complete tumor regression in an animal model of cancer in combination with anticancer therapy, such as radiotherapy;

(ix) shows at least a 60% reduction in tumor growth in a syngeneic tumor xenograft model, for example, when administered at a dose of ≤10 mg/kg;

(x) enhances an antitumor response in the presence of one or more immunomodulators, e.g., a checkpoint inhibitor antagonist, e.g., a PD-1 or CTLA-4 antagonist, or an immune response activator, e.g., a 4-1BB agonist, when administered to a subject, e.g., a mouse or person;

(xi) has an efficacy that is independent of αvβ8 integrin expression in a tumor model;

(xii) increases the abundance of CD8+ GzmB+ T cells in the tumor microenvironment, for example, as monotherapy;

(xiii) shows a reduction, e.g., at least an 80% reduction in tumor growth when used in combination with a checkpoint inhibitor antagonist (e.g., anti-PD-1 or anti-PD-L1 antibody), e.g., in a syngeneic model of squamous cell carcinoma, cancer breast and/or colon cancer;

(xiv) demonstrates a statistically significant improvement in overall survival of the subject, e.g., human or mouse, as determined by Kaplan-Meier analysis;

(xv) has a high degree of thermal stability;

(xvi) shows minimal aggregation at high concentrations; And

(xvii) can demonstrate reproducible expression and purity under industrial production conditions.

E186. The method of embodiment E184 or E185, wherein said antibody or antigen binding fragment thereof has at least one of the following properties:

(i) a binding affinity, expressed as KD, for human αvβ8 integrin that is less than the KD for mouse ADWA11 antibody, eg, less than 536 pM;

(ii) a KD for human αvβ8 integrin that is less than or equal to 100 pM for purified human αvβ8 integrin;

(iii) KD for mouse αvβ8 integrin, which is less than 100 pM;

(iv) KD for cynomolgus αvβ8 integrin, which is less than 100 pM;

(v) KD for rat αvβ8 integrin, which is approximately 160 pM;

(vi) shows approximately equivalent affinities for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, eg, with a KD that is less than 100 pM, eg, determined using the Biacore affinity assay.

(vii) IC50 for inhibition of TGFβ transactivation, which is less than 183 pM;

(viii) IC50 for inhibition of TGFβ transactivation in U251 cells approximately 199 +/- 93.6 pM;

(ix) IC50 for inhibition of TGFβ transactivation, which is from about 100 pM to about 300 pM.

(x) EC50 for U251 cells approximately 126 pM with standard deviation plus/minus 34 pM;

(xi) EC50 for U251 cells is approximately 256 pM with a standard deviation of plus/minus 115 pM;

(xii) EC50 for U251 cells from about 80 pM to about 400 pM;

(xiii) EC50 for C8-S cells approximately 115 pM;

(xiv) EC50 for C8-S cells is approximately 145 pM with a standard deviation of plus/minus 23.7 pM;

(xv) EC50 for C8-S cells from about 110 pM to about 180 pM;

(xvi) at least one predicted human pharmacokinetic (PK) parameter selected from:

A. clearance from the central chamber (CL) is approximately 0.12-0.15 ml/h/kg;

b. intercompartmental clearance distribution (CLF) approximately 0.15-0.51 ml/h/kg;

With. volume of distribution for the central chamber (V1) approximately 36-39 ml/kg;

d. volume of distribution for the peripheral chamber (V2) approximately 21-33 ml/kg; and/or

e. the final half-life (t _1/2 ) is approximately 12-17 days; And

(xvii) does not show detectable binding to human Fcγ or C1q receptors.

E187. The method of any one of embodiments E184-E186, wherein the antibody or antigen-binding fragment thereof corresponds to any of embodiments E1-E166, or the pharmaceutical composition of embodiment E167.

E188. The method of any one of embodiments E184-E187, wherein the antibody or antigen binding fragment thereof is administered in an amount sufficient to increase the infiltration of CD45+ cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and/or cellular expression of Granzyme B.

E189. The method of any one of embodiments E184-E188, wherein the antibody or antigen binding fragment thereof is administered in an amount sufficient to enhance CD8+ T cell infiltration.

E190. The method of any one of embodiments E184-E189, wherein the antibody or antigen binding fragment thereof is administered in an amount sufficient to increase expression of Granzyme B on CD8+ T cells.

E191. The method of any one of embodiments E184-E190, wherein the antibody or antigen binding fragment thereof is administered in an amount sufficient to increase the accumulation of inflammatory macrophages having an increased level of Ly6G expression.

E192. The method of any one of embodiments E184-E191, wherein the antibody or antigen-binding fragment thereof is administered in an amount sufficient to increase the accumulation of CD45+CD11b+CD11c-Ly6G-Ly6C ^high CD206 ^low inflammatory macrophages.

E193. The method of any one of embodiments E184-E192, wherein the antibody or antigen binding fragment thereof is administered in an amount sufficient to enhance the response to the second therapy.

E194. The method of any one of embodiments E184-E193, wherein the effectiveness of the antibody or antigen binding fragment thereof when administered to an animal tumor model is independent of expression of αvβ8 integrin by the tumor model.

E195. The method according to any one of embodiments E184-E194, wherein the administration of said antibody or antigen-binding fragment thereof occurs in combination with a second therapy.

E196. The method of embodiment E195, wherein the second therapy includes an anticancer therapy, a cytotoxic or cytostatic agent, such as a chemotherapeutic agent, hormonal treatment, a vaccine, and/or an immunotherapy.

E197. The method of embodiment E195 or E196, wherein the second therapy is or includes surgery, radiation, cryosurgery, and/or thermotherapy.

E198. The method of any one of embodiments E195-E197, wherein the second therapy comprises a modulator, e.g., an inhibitor or agonist, of an immune checkpoint molecule, optionally wherein the second therapy is or contains an immune checkpoint molecule modulator selected from the group consisting of PD1, PD-L1, 4-1BB, OX40, CTLA-4, PD-L2, TIM-3, LAG-3, VISTA, CD160, BTLA, TIGIT, 2B4, TGFβ, LAIR1 and combinations thereof.

E199. The method of embodiment E198, wherein the immune checkpoint molecule inhibitor is a PD1, PD-L1, CTLA-4, PD-L2, TIM-3, LAG-3, VISTA, BTLA, TIGIT, 2B4, TGFβ, or LAIR1 inhibitor.

E200. The method of embodiment E199, wherein the immune checkpoint molecule inhibitor is a PD1 inhibitor, for example, an anti-PD1 antibody.

E201. The method of embodiment E199, wherein the immune checkpoint molecule inhibitor is a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody or a soluble CTLA-4 fusion.

E202. The method of embodiment E195-E201, wherein the second therapy contains a costimulatory molecule agonist.

E203. The method of embodiment E202, wherein the costimulatory molecule agonist is selected from at least one of 4-1BB (CD137), OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160 or B7-H3.

E204. The method of embodiment E202, wherein the agonist of the costimulatory molecule is a 41-BB agonist.

E205. The method of embodiment E195-E204, wherein the second therapy contains a PARP1 inhibitor (e.g., olaparib, rucaparib, niraparib, veliparib, iniciarib, talazoparib, 3-aminobenzamide CEP 9722, E7016, BSI-201, KU-0059436, AG014699, MK-4827 or BGB-290).

E206. The method of embodiment E184-E205, wherein the cancer is selected from the group consisting of solid tumor, hematologic cancer ( eg , leukemia, lymphoma, myeloma, eg , multiple myeloma) and metastatic sites.

E207. The method of embodiment E206, wherein the cancer is a solid tumor.

E208. The method of embodiment E206 or E207, wherein the cancer is a solid tumor and is selected from malignancies, e.g. , sarcomas and carcinomas, e.g. , adenocarcinomas of various organ systems, e.g., lung ( e.g. , non-small cell lung cancer (NSCLC)), breast, ovary , lymphoid, gastrointestinal tract ( eg colon), rectum, genital and genitourinary tract ( eg kidney, urothelial, bladder cells, prostate), pharynx, central nervous system ( eg brain, nerve or glial cells), head and neck ( eg , squamous cell carcinoma (HNSCC), head and neck, skin ( eg , melanoma, eg , advanced melanoma), pancreas, colon, rectal, kidney (eg, renal cell carcinoma), liver, small bowel cancer and esophageal cancer, stomach and esophageal cancer, thyroid cancer and cervical cancer.

E209. The method of any one of embodiments E184-E208, wherein the cancer is a lymphoproliferative disease ( eg , post-transplant lymphoproliferative disease) or hematologic cancer, T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or leukemia ( eg , myeloid leukemia or lymphoid leukemia) .

E210. The method of any one of embodiments E184-E209, wherein the cancer is early, intermediate or late stage or metastatic cancer.

E211. The method of any one of embodiments E184-E210, wherein the cancer is selected from the group consisting of renal cell carcinoma, ovarian cancer, and squamous cell carcinoma of the head and neck.

E212. The method of embodiment E211 further comprising administering a checkpoint inhibitor, such as a PD-1, PD-L1, or CTLA-4 inhibitor.

E213. The method of embodiment E212, wherein the PD-L1 inhibitor is not avelumab.

E214. The method of embodiment E211 or E212, wherein the cancer is kidney cancer, eg , renal cell carcinoma (RCC).

E215. The method of embodiment E214, wherein the cancer is renal cancer selected from the group consisting of metastatic RCC, clear cell renal cell carcinoma (ccRCC), non-clear cell renal cell carcinoma (ncRCC), and high-risk renal cell carcinoma.

E216. The method of embodiment E215, wherein the antibody or antigen binding fragment thereof is administered as 1st line or 2nd line therapy.

E217. The method of any one of embodiments E184-E216, wherein the antibody or antigen-binding fragment thereof is administered as first-line therapy.

E218. The method according to any one of embodiments E184-E216, wherein the antibody or antigen-binding fragment thereof is administered as a 2nd line therapy.

E219. The method of any one of embodiments E184-E212, wherein the cancer is ovarian cancer.

E220. The method of embodiment E219, wherein the second therapy is a PARP1 inhibitor (e.g., olaparib, rucaparib, niraparib, veliparib, iniparib, talazoparib, 3-aminobenzamide, CEP 9722, E7016, BSI-201, KU-0059436, AG014699, MK-4827, or BGB-290).

E221. The method of embodiment E219, wherein the antibody or antigen binding fragment thereof is administered as a 2nd line therapy, optionally, where the subject has platinum resistance.

E222. The method of embodiment E219, wherein the antibody or antigen binding fragment thereof is administered as 1st line therapy.

E223. The method of any one of embodiments E184-E222, wherein the cancer is squamous cell carcinoma of the head and neck.

E224. The method of embodiment E223, where the method further comprises administering radiation therapy.

E225. The method of embodiment E223 or E224, wherein the cancer is platinum-resistant and/or recurrent cancer.

E226. The method of any one of embodiments E179-E225, wherein said subject is a human.

E227. The method of any one of embodiments E179-E226, comprising administering said antibody or antigen-binding fragment thereof or pharmaceutical composition intravenously.

E228. The method according to any one of embodiments E179-E227, comprising administering said antibody or antigen-binding fragment or pharmaceutical composition subcutaneously.

E229. The method of any one of embodiments E179-E228, wherein said antibody or antigen binding fragment or pharmaceutical composition thereof is administered approximately twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months or once every twelve months.

E230. The method of any one of embodiments E179-E229, wherein the antibody or antigen-binding fragment thereof is administered every two weeks, for example, up to 12 times (for example, up to 10, 8, 6, 5, 4 or 3 times).

E231. The method of embodiment E230, wherein each administration contains 5-10 mg/kg (eg, 5, 6, 7, 8, 9 or 10 mg/kg) of antibody or antigen binding fragment thereof.

E232. The method of embodiment E231, where each administration contains approximately 7 mg/kg.

E233. The method of any one of embodiments E179-E229, wherein the antibody or antigen-binding fragment thereof is administered every four weeks, for example, up to 6 times (for example, up to 6, 5, 4, 3, 2 or 1 time).

E234. The method of embodiment E233, wherein each administration contains 10-15 mg/kg (eg, 10, 11, 12, 13, 14, or 15 mg/kg) of antibody or antigen-binding fragment thereof.

E235. The method of embodiment E234, where each administration contains approximately 12 mg/kg.

E236. A method for detecting αvβ8 integrin (e.g., human αvβ8 integrin) in a sample, tissue, or cell using the antibody or antigen-binding fragment thereof of any one of Embodiments E1-E166 or the pharmaceutical composition of Embodiment E167, comprising contacting the sample, tissue, or cell with the antibody and antibody definitions.

E237. A kit containing an antibody or antigen binding fragment thereof of any one of embodiments E1-E166 or a pharmaceutical composition of embodiment E167.

E238. The antibody or antigen binding fragment thereof of any one of embodiments E1-E166 or the pharmaceutical composition of embodiment E167, for use as a drug, for example, in any of the method embodiments described herein.

E239. An isolated antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin, where the antibody or fragment is at least one antibody or fragment selected from the group consisting of:

(a) an antibody or antigen-binding fragment thereof containing complementarity determining region light chain 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO:11; CDR-L2 containing the amino acid sequence of SEQ ID NO:12; CDR-L3 containing the amino acid sequence of SEQ ID NO:13; Heavy chain CDR1 (CDR-H1) containing the amino acid sequence of SEQ ID NO:8; CDR-H2 containing the amino acid sequence of SEQ ID NO:9; and CDR-H3 containing the amino acid sequence of SEQ ID NO:10;

(b) an antibody or antigen-binding fragment thereof comprising a CDR-L1 containing the amino acid sequence of SEQ ID NO:17; CDR-L2 containing the amino acid sequence of SEQ ID NO:18; CDR-L3 containing the amino acid sequence of SEQ ID NO:19; CDR-H1 containing the amino acid sequence of SEQ ID NO:14; CDR-H2 containing the amino acid sequence of SEQ ID NO:15; and CDR-H3 containing the amino acid sequence of SEQ ID NO:16;

(c) an antibody or an antigen-binding fragment thereof comprising a light chain variable region (VL) containing the amino acid sequence encoded by the plasmid insert deposited with ATCC and having accession number PTA-124918, and a heavy chain variable region (VH) containing the amino acid sequence, encoded by the plasmid insert deposited at the ATCC with accession number PTA-124917;

(d) an antibody or antigen-binding fragment thereof comprising a VL region containing the amino acid sequence of SEQ ID NO:7 and a VH region containing the amino acid sequence of SEQ ID NO:6;

(e) an antibody or an antigen-binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:62-66, and a VH region containing an amino acid sequence selected from the group consisting of SEQ ID NO:34 -38;

(f) an antibody or antigen binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:47 and 92, and a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:39 and 88-91;

(g) an antibody or antigen-binding fragment thereof comprising a VL region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 67-69, and a VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs :6 and 93;

(h) an antibody or antigen binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 47-69 and 92, and a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, 34-46, 88-91 and 93;

(i) an antibody or an antigen binding fragment thereof comprising a light chain (LC) region containing the amino acid sequence of SEQ ID NO:5 and a heavy chain (HC) region containing the amino acid sequence of SEQ ID NO:2;

(j) an antibody or an antigen-binding fragment thereof comprising an LC region containing the amino acid sequence of SEQ ID NO:5, and an HC region containing the amino acid sequence of SEQ ID NO:3;

(k) an antibody or an antigen-binding fragment thereof comprising an LC region containing the amino acid sequence of SEQ ID NO:123, and an HC region containing the amino acid sequence of SEQ ID NO:124 or 182;

(l) an antibody or an antigen-binding fragment thereof comprising a VL region encoded by the nucleic acid sequence SEQ ID NO:186 and a VH region encoded by the nucleic acid sequence SEQ ID NO:190; And

(m) an antibody or an antigen-binding fragment thereof comprising an LC region encoded by the nucleic acid sequence SEQ ID NO:185 and an HC region encoded by the nucleic acid sequence SEQ ID NO:189 or 191.

E240. The isolated antibody or antigen binding fragment thereof of embodiment E239, comprising a VL region containing the amino acid sequence of SEQ ID NO:7, and a VH region containing the amino acid sequence of SEQ ID NO:6.

E241. An isolated antibody or antigen binding fragment thereof from embodiments E239 or E240, comprising a VL region containing an amino acid sequence at least 95% identical to SEQ ID NO:7, and a VH region containing an amino acid sequence at least 95% identical to SEQ ID NO:6 .

E242. An isolated antibody or antigen binding fragment thereof from embodiment E239, comprising an LC region containing the amino acid sequence of SEQ ID NO:5, and an HC region containing the amino acid sequence of SEQ ID NO:2 or 3.

E243. An isolated antibody or antigen binding fragment thereof from embodiments E239 or E242, comprising an LC region containing an amino acid sequence at least 95% identical to SEQ ID NO:5, and an HC region containing an amino acid sequence at least 95% identical to SEQ ID NO:2 or 3.

E244. An isolated antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin, wherein the antibody or fragment comprises a VH region containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 34-46, 88-91 and 93, and/or a VL region containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO : 7, 47-69 and 92.

E245. An isolated antibody or antigen-binding fragment thereof that specifically binds the αvβ8 integrin, wherein the antibody or fragment comprises:

(i) an HC antibody containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2 or 3; and/or

(ii) an LC antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5.

E246. An isolated antibody that specifically binds the αvβ8 integrin, comprising an LC consisting of the amino acid sequence of SEQ ID NO:5 and an HC consisting of the amino acid sequence of SEQ ID NO:2 or 3.

E247. An isolated antibody that specifically binds αvβ8 integrin, containing:

a VL region of an antibody containing CDR-L1, CDR-L2 and CDR-L3 from the VL region containing the amino acid sequence of SEQ ID NO:7; And

VH region of an antibody containing CDR-H1, CDR-H2 and CDR-H3 from the VH region containing the amino acid sequence SED ID NO:6.

E248. The isolated antibody of embodiment E247 comprising an antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 181 or 184 and an antibody light chain constant region comprising the amino acid sequence of SEQ ID NO: 83.

E249. An isolated antibody that specifically binds αvβ8 integrin, containing:

a) a VL region of an antibody containing the first, second and third CDRs of the VL region containing the amino acid sequence of SEQ ID NO:7;

a VH region of an antibody comprising the first, second and third CDRs of the VH region containing the amino acid sequence of SEQ ID NO:6;

an antibody light chain constant region (CL) comprising the amino acid sequence of SEQ ID NO:83; And

an antibody heavy chain constant region (CH) comprising the amino acid sequence of SEQ ID NO:181 or 184;

b) a VL region of an antibody containing an amino acid sequence at least 95% identical to SEQ ID NO: 7; and a VH region of the antibody containing an amino acid sequence at least 95% identical to SEQ ID NO:6; or

c) an antibody LC region containing an amino acid sequence at least 95% identical to SEQ ID NO: 5, and an antibody HC region containing an amino acid sequence at least 95% identical to SEQ ID NO: 2 or 3.

E250. An isolated antibody or antigen-binding fragment thereof that specifically binds the αvβ8 integrin, comprising an antibody VH containing the amino acid sequence encoded by the insert deposited in the ATCC and having accession number PTA-124917, and an antibody VL containing the amino acid sequence encoded by the insert deposited in the ATCC and having access number PTA-124918.

E251. An isolated antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin, where the antibody or fragment has at least one of the following properties:

A. a binding affinity, expressed as KD, for human αvβ8 integrin that is less than the KD for mouse ADWA11 antibody, for example, less than about 536 pM;

b. KD for human αvβ8 integrin, which is less than or equal to about 100 pM;

With. a KD for mouse αvβ8 integrin that is less than the KD for mouse ADWA11 antibody, for example, less than about 489 pM;

d. KD for mouse αvβ8 integrin, which is less than about 100 pM;

e. a KD for cynomolgus αvβ8 integrin that is less than the KD for mouse ADWA11 antibody, for example, less than about 507 pM;

f. KD for cynomolgus αvβ8 integrin, which is less than or equal to approximately 100 pM;

g. KD for rat αvβ8 integrin, which is approximately 160 pM;

h. approximately equivalent affinities for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, for example, with a KD that is less than about 100 pM, determined using the Biacore affinity assay;

i. IC50 for inhibition of TGFβ transactivation, which is from about 100 pM to about 300 pM;

j. EC30 for U251 cells from about 100 pM to about 400 pM;

k. EC50 for C8-S cells from about 110 pM to about 180 pM; And

l. at least one predicted human pharmacokinetic (PK) parameter selected from:

i. clearance from the central chamber (CL) is approximately 0.12 ml/h/kg;

ii. intercompartmental clearance distribution (CLF) approximately 0.51 ml/h/kg;

iii. volume of distribution for the central chamber (V1) approximately 36 ml/kg;

iv. volume of distribution for the peripheral chamber (V2) approximately 33 ml/kg;

v. the final half-life (t _1/2 ) is approximately 15-17 days; And

vi. lack of detectable binding to human Fcγ receptors or C1q.

E252. An isolated antibody or antigen binding fragment thereof according to any of the above embodiments, comprising a human IgG1 Fc region containing one or more substitutions selected from positions L234, L235 and G237 (for example, one or more of L234A, L235A and G237A), numbered according to Eu numbering according to Kabat.

E253. An isolated antibody or antigen binding fragment thereof from any of the above embodiments, wherein the antibody is a humanized antibody, a human antibody, a mouse antibody, a chimeric antibody, or a camel antibody.

E254. An isolated antibody or antigen-binding fragment thereof from any of the above embodiments, wherein the antibody heavy chain isotype is selected from IgG1, IgG2, IgG3, IgG4, or any variant thereof; and/or wherein the light chain constant region is selected from kappa or lambda.

E255. An isolated antibody or antigen binding fragment thereof from any of the above embodiments, wherein the heavy chain isotype of the antibody is IgG1 and/or where the light chain constant region is a kappa light chain.

E256. An antibody or antigen binding fragment thereof that competes for binding to αvβ8 integrin with the antibody or antigen binding fragment thereof of embodiment E242.

E257. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof from any of the above embodiments and a pharmaceutically acceptable carrier or excipient.

E258. The pharmaceutical composition of embodiment E257, comprising i) an antibody or an antigen-binding fragment thereof comprising a heavy chain encoded by the amino acid sequence of SEQ ID NO:2 and an antibody light chain encoded by the amino acid sequence of SEQ ID NO:5, ii) an antibody or an antigen-binding fragment thereof comprising an antibody heavy chain encoded by the amino acid sequence SEQ ID NO:3 and an antibody light chain encoded by the amino acid sequence SEQ ID NO:5 or iii) both.

E259. An isolated nucleic acid molecule that encodes an antibody or antigen binding fragment thereof according to any one of embodiments E239-E256.

E260. The isolated nucleic acid of embodiment E259, wherein the isolated nucleic acid encodes a VH region, a VL region, or both of an antibody or an antigen binding fragment thereof, and wherein said nucleic acid comprises: a nucleic acid sequence of SEQ ID NO:190, a nucleic acid sequence of SEQ ID NO:186 or both.

E261. The isolated nucleic acid of embodiment E259, wherein the isolated nucleic acid encodes a heavy chain constant region, a light chain constant region, or both of an antibody or an antigen binding fragment thereof, and wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 192 or 193; nucleic acid sequence SEQ ID NO: 194; or both.

E262. The isolated nucleic acid of embodiment E259, wherein the isolated nucleic acid encodes an HC, LC, or both of an antibody or an antigen binding fragment thereof, and wherein said nucleic acid comprises: a nucleic acid sequence of SEQ ID NO:189 or 190; nucleic acid sequence SEQ ID NO185; or both.

E263. The isolated nucleic acid of embodiment E259, wherein the isolated nucleic acid contains the nucleic acid sequence of interest of a plasmid deposited with the ATCC and having accession number PTA-124917, the nucleic acid sequence of interest of a plasmid deposited with the ATCC and having accession number PTA -124918, or both.

E264. The isolated nucleic acid of embodiment E259, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 85%, 87%, 90%, 92%, 93%, 95%, 97%, 98%, or 100% identity sequences with SEQ ID NO: 189 or SEQ ID NO: 191; a nucleic acid sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% sequence identity to SEQ ID NO: 185; or both.

E265. A vector containing the nucleic acid of any one of embodiments E259-E264.

E266. A host cell containing the nucleic acid of any one of embodiments E259-E264 or the vector of embodiment E265.

E267. The host cell of embodiment E265, wherein the host cell is a mammalian cell selected from the group consisting of CHO cells, COS cells, HEK-293 cells, NS0 cells, PER.C6® cells, and Sp2.0 cells.

E268. A method of producing an isolated antibody or antigen-binding fragment thereof, comprising culturing the host cell of Embodiment 266 under conditions where the antibody or fragment is expressed by the host cell, and isolating the antibody or fragment.

E269. A method of reducing αvβ8 integrin activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof of any one of embodiments E239-E256, or a pharmaceutical composition of embodiments E257 or E258.

E270. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof of any one of embodiments E239-E256, or a pharmaceutical composition of embodiment E257 or E258.

E271. The method of embodiment E270, further comprising the use of a cytotoxic agent, a cytostatic agent, a chemotherapeutic agent, hormonal treatment, a vaccine, immunotherapy, surgery, radiation, cryosurgery, thermotherapy, or a combination thereof.

E272. The method of embodiment E271, wherein the additional administration is carried out simultaneously, sequentially or separately from the administration of a therapeutically effective amount of the antibody or antigen binding fragment or pharmaceutical combination thereof.

E273. The method of embodiment E271, wherein the immunotherapy comprises an immune checkpoint molecule modulator selected from the group consisting of anti-PD1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, soluble CTLA- 4 fusion protein or combination, and where the anti-PD-L1 antibody is not avelumab.

E274. The method of any one of embodiments E270-E273, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma, clear cell or papillary renal cell carcinoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, gastroesophageal junction cancer , esophageal cancer, squamous cell lung cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, uterine cancer, melanoma, urothelial carcinoma and combinations thereof.

E275. A method for detecting αvβ8 integrin in a sample, tissue, or cell using an antibody or an antigen-binding fragment thereof of embodiments E239-E256, comprising contacting the sample, tissue, or cell with the antibody or an antigen-binding fragment thereof and detecting the antibody or antigen-binding fragment thereof.

E276. A kit comprising an antibody or fragment of any of embodiments E239-E256 or a pharmaceutical composition of embodiments E257 or E258 and optionally containing a modulator of embodiment E273.

E277. The antibody or antigen binding fragment thereof of any one of embodiments E239-E256 or a pharmaceutical composition of embodiments E257 or E258 for use in reducing αvβ8 integrin activity in a subject in need thereof for the treatment of cancer.

E278. The antibody or antigen binding fragment thereof of any one of embodiments E239-E256, or the pharmaceutical composition of embodiments E257 or E258 for use in the treatment of cancer, optionally wherein the antibody or antigen binding fragment thereof or pharmaceutical composition is intended to be administered simultaneously, sequentially or separately to combinations with immunotherapy, where the combination does not necessarily have a synergistic therapeutic effect.

E279. An antibody or antigen-binding fragment thereof or a pharmaceutical composition for use in embodiment E278, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma, clear cell or papillary type renal cell carcinoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer , esophageal junction cancer, esophageal cancer, squamous cell lung cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, uterine cancer, melanoma, urothelial carcinoma, and combinations thereof, optionally, wherein the antibody or antigen-binding fragment thereof or pharmaceutical composition or combination is intended for use in conjunction with the administration of immunotherapy or radiation therapy.

E280. Use of an antibody or antigen binding fragment thereof of any one of embodiments E239-E256 or a pharmaceutical composition of embodiments E257 or E258 for the treatment of cancer.

E281. Use of the antibody or antigen binding fragment thereof of any one of embodiments E239-E256 in the manufacture of a medicament for the treatment of cancer.

E282. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of (i) an antibody or antigen binding fragment thereof that specifically binds αvβ8 integrin, and (ii) an anti-PD1, anti-PD-L1 or anti-PD-L2 modulator immune checkpoint molecules.

E283. The method of embodiment E282, wherein the cancer is squamous cell carcinoma.

E284. The method of embodiment E282, wherein the cancer is breast cancer or colon cancer.

E285. The method as in any one of embodiments E282-E284, wherein the modulator is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, and an anti-PD-L2 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary as well as the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, the following drawings show embodiments, however, it should be understood that the invention is not limited to the precise circuits and tools shown.

In FIG. 1A shows a sequence alignment comparing the heavy chain variable regions of the amino acid sequences of the mouse hybridoma ADWA11 antibody (referred to as "mADWA11", "ADWA11" or "ADWA11 mouse hybridoma"; SEQ ID NO:20), humanized ADWA11 VH05-2/VK01(2.4) antibody (“huADWA11-2.4”, “ADWA11 2.4” or “humanized ADWA11-2.4”; SEQ ID NO:6) and germline IGHV3-07 (“IMGT” or “DP-54”; SEQ ID NO:195) sequences. The underlined amino acid residues are CDR sequences according to Kabat.

In FIG. 1B shows a sequence alignment comparing the light chain variable regions of the amino acid sequences of the mouse hybridoma ADWA11 antibody (referred to as "mADWA11", "ADWA11" or "ADWA11 mouse hybridoma"; SEQ ID NO:21), humanized ADWA11 VH05-2/VK01(2.4) antibody (“huADWA11-2.4”, “ADWA11 2.4” or “humanized ADWA11 2.4”; SEQ ID NO:7) and germline IGKV1-39 (“IMGT” or “DPK-9”; SEQ ID NO:196) sequences. The underlined amino acid residues are CDR sequences according to Kabat.

In FIG. 2 shows a representative graph comparing the binding specificity of mouse hybridoma antibodies ADWA2 and ADWA11 to the human integrins αvβ3 (“AVB3”) and αvβ6 (“AVB6”) by ELISA. ADWA2 and ADWA11 do not bind αvβ3 (“AVB3”) and αvβ6 (“AVB6”) integrins, while the control αV binding antibody (“AlphaV mAb”) binds both αvβ3 and αvβ6.

In FIG. 3A is a representative graph demonstrating that mouse hybridoma ADWA11 antibody binds to human αvβ8 by ELISA.

FIG. 3B is a representative graph showing that the humanized ADWA11 antibody VH05/VK01(2.4) binds to human αvβ8 by ELISA.

In FIG. 4A is a representative graph showing the ELISA binding affinity of ADWA11 VH05/VK01 Fabs having K30A, N55Q, N57Q, D61E, P62A, or K63A amino acid substitutions in the heavy chain variable region to human αvβ8, compared with the binding affinity of the parent humanized ADWA11_VH05/VK01 (ADWA VH_1.5 and ADWA VL_1.1) antibodies with human αvβ8. Fabs having a K30A, N55Q, N57Q, D61E, P62A, or K63A amino acid substitution in the heavy chain variable region retain binding affinity for human αvβ8.

In FIG. 4B is a representative graph showing the ELISA binding affinity of ADWA11 VH05-2/VK01 Fabs having an amino acid substitution in either the heavy chain variable region (e.g., F64V) or the light chain variable region (e.g., L30S, Y55A, A60Q , M94Q, L97Y, F101L, F101W, or Q105G) to human αvβ8, compared with the binding affinity of the parent ADWA11 VH05-2/VK01 antibody to human αvβ8. Fabs having a Y55A, A60Q, F101L or F101W amino acid substitution in the light chain variable region exhibit reduced binding affinity to human αvβ8 compared to the humanized ADWA11 VH05-2/VK01 antibody. Other Fabs tested retained binding affinity for human αvβ8.

In FIG. 4C shows representative graphs demonstrating the ELISA binding affinity of ADWA11 VH05-2/VK01 Fab, referred to as VH05-2/VK01(2.1) (ADWA11 2.1), VH05-2/VK01(2.2) (ADWA 2.2 ), VH05-2/VK01(2.3) (ADWA 2.3), VH05-2/VK01(2.4) (ADWA 2.4), VH05-2(F64V)/VK01(2.1), VH05-2(F64V)/VK01 (2.3) and VH05-2(F64V)/VK01(2.4) having the combination of amino acid substitutions shown in Table 5 with human αvβ8 and compared to the parent antibody (VH05-2_VK01 parent). Each of the Fabs tested retained binding affinity for human αvβ8.

In FIG. 5 shows a representative graph comparing the binding specificity of mouse hybridoma ADWA11 (“MsADWA11” or “mADWA11”) and humanized ADWA11 VH05-1/VK01 (“ADWA11 5-1_1”) and ADWA11 VH05-2/VK01 (“ADWA11 5-2_1”) for human integrins αvβ3 (avb3) and αvβ6 (avb6), according to Biacore. ADWA11 antibodies do not bind αvβ3 (avb3) and αvβ6 (avb6) integrins, while the control αV binding antibody (“anti-av”) binds both αvβ3 and αvβ6.

In FIG. 6A shows representative binding traces of Biacore for ADWA11 hybridoma (“Ms ADWA11”) Fab to human αvβ8.

In FIG. 6B shows representative Biacore binding traces for a humanized ADWA11_5-2 2.4 Fab, also referred to as ADWA11VH05-2/VK01(2.4) or “ADWA11 2.4” Fab, to human αvβ8.

In FIG. 6C is a representative table demonstrating that the humanized Fab ADWA11, referred to herein as ADWA115-2 2.4 (also known as ADWA11 2.4 and ADWA11 VH05-2/VK01(2.4)), retains affinity for human αvβ8 and cross-species reactivity as reported Biacore, compared to the parent Fab mouse antibody (“MsADWA11”). ADWA11 5-2 2.4 shows equivalent affinity for human, cynomolgus, mouse and rat αvβ8 with a KD <200 pM. The parent mouse antibody shows equivalent affinity for human, cynomolgus and mouse αvβ8 with a KD of 489-536 pM.

In FIG. 7A shows a representative graph comparing cell U251 binding data for ADWA11 VH05-2/VK01 (parent) Fab (“ADWA11”) having a single amino acid substitution in either the heavy chain variable region (eg, F64V) or the light chain variable region (eg , L30S, M94Q, L97Y, F101L or Q105G). Fabs having the F101L amino acid substitution in the light chain variable region show reduced binding to U251 cells (human αvβ8) compared to the parent antibody. Other Fabs tested retained binding to U251 cells (human αvβ8).

In FIG. 7B shows a representative graph comparing cell U251 binding data for ADWA11 VH05-2/VK01 (parent) Fabs (“ADWA11”) having a single amino acid substitution in the light chain variable region (eg, M56A or N58S). M56A and N58S Fab retained binding to U251 cells (human αvβ8).

In FIG. 7C is a representative graph showing U251 cell binding data for ADWA11 VH05-2/VK01 Fab (“ADWA11”) having a combination of amino acid substitutions termed 2.1, 2.2, 2.3, 2.4, 2.1 (F64V), 2.3 (F64V) and 2.4 ( F64V) according to table 5, compared with the original antibody. Each of the Fabs tested retained binding to U251-fixed cells.

In FIG. 8 shows a representative graph comparing the binding affinities of ADWA11 mIgG_4mut and ADWA11 VH05/VK01 antibodies to U251 cells.

In FIG. 9A shows representative graphs demonstrating the binding of ADWA11 VH05-2/VK01 having a combination of amino acid substitutions called ADWA11_VH05-2/VK01(2.1) and ADWA11_VH05-2/VK01(2.4) to U251 (human glioblastomas) or C8-S (mouse astrocytes ) cells. ADWA11 VH05-2/VK01(2.1) and ADWA11 VH05-2/VK01(2.4) antibodies retained binding to U251 (human avb8) and C8-S (mouse avb8) cells.

In FIG. 9B shows representative graphs depicting the binding of integrin-specific antibodies such as ADWA11 VH05-2/VK01(2.4) to HEK cells expressing αvβ3, αvβ5, αvβ6 and αvβ8. Results show saturable binding of ADWA11 VH05-2/VK01(2.4) to HEK cells expressing αvβ8 and lack of binding to cells expressing αvβ3, αvβ5, αvβ6. These results demonstrate the specific binding of ADWA11 VH05-2/VK01(2.4) to human αvβ8.

In FIG. 10A shows a representative graph comparing the effect of ADWA11 mouse hybridoma (“mFab”) and humanized ADWA11 Fab: ADWA11 VH01/VK01, ADWA11 VH02/VK01, ADWA11 VH02/VK02, ADWA11 VH05/VK02 and ADWA11 VH05/VK01 on TGFβ trans-activation by U251 cells . VH01/VK01, VH02/VK01, and VH02/VK02 Fabs show reduced activity, while VH05/VK01 retains activity and VH05/VK02 shows improved TGFβ activation blocking activity in a U251 transactivation assay compared to a mouse hybridoma (“mFab”) ADWA11 Fab ).

In FIG. 10B shows a representative graph comparing the effects of specified ADWA11 VH05-2/VK01 Fabs having an amino acid substitution in either the heavy chain variable region (e.g., F64V) or the light chain variable region (e.g., L30S, M94Q, L97Y, F101L, F101W, or Q105G ) on TGFβ transactivation in U251 cells. Fabs having an F101L or F101W single amino acid substitution in the light chain variable region exhibit reduced effects on TGFβ transactivation compared to the humanized parent ADWA11 VH05-2/VK01 Fab. Other Fabs tested retain activity in the TGFβ transactivation assay.

In FIG. 10C shows a representative graph comparing the effects of ADWA11 VH05-2/VK01 Fabs having amino acid substitutions, including a combination of amino acid substitutions referred to as 2.1, 2.2, 2.3, 2.4, 2.1 (F64V), 2.3 (F64V) and 2.4 (F64V) according to Table 5. VH02-2/VK01(2.3) and VH05-2(F64V)/VK01(2.3) Fabs exhibit reduced effects on TGFβ transactivation compared to the parent ADWA11 VH05-2/VK01 Fab. Other Fabs tested retain activity in the TGFβ transactivation assay.

In FIG. 10D shows a representative graph comparing the effect of ADWA11 VH05-2/VK01 (parent) Fabs having the indicated amino acid substitution in the heavy chain variable region (e.g., F64V) or light chain variable region (e.g., L30S, M94Q, L97Y, Q105G, M56A, or N58S ). The tested Fabs retained activity in the TGFβ transactivation assay compared to the parent ADWA11 VH05-2/VK01 Fab.

In FIG. 10E is a representative graph showing the effect of humanized ADWA11 VH05/VK01, VH05/VK01-D61E (VH05-1/VK01) and VH05/VK01-N55Q-D61E (VH05-2/VK01) IgG on TGFβ transactivation by U251 cells. The antibodies tested remained active in the TGFβ transactivation assay.

In FIG. 10F shows representative graphs demonstrating the effect of ADWA11_VH05-2_VK01(2.4) on TGFβ transactivation by U251 (left panel) and C8-S (right panel) cells compared to isotype control antibody. Additional experiments demonstrated an IC50 for ADWA11 VH05-2/VK01(2.4) in a TGFβ transactivation assay with U251 cells of 199 ± 93.6 pM (mean ± standard deviation).

In FIG. 11 is a representative graph showing the proportion of responders (antigenicity) for various ADWA11 VH05-2VK01 CDR peptides compared to the positive control peptides listed in Table 1. The peptide antigenicity assessment is used to select candidate CDR sequences with a reduced risk of immunogenicity.

In FIG. 12A shows representative graphs demonstrating the effectiveness of combination treatment of anti-αvβ8 (ADWA11), anti-PD1 antibody (“PD-1”, RMP1-14), mIgG1_4mut isotype (2B8) and rat IgG2a isotype (2A3) in the EMT6 model of breast cancer. Tumor growth is measured using digital calipers three times a week and shown as tumor volume (length x width x width x 0.5). The average tumor volume +/- SEM in each treatment group is plotted until less than 8/10 mice remain in each group. Survival is defined as the time to reach 1000 mm ³ . The combination of anti-PD1 and ADWA11 inhibits tumor growth and improves overall survival to a greater extent than other combinations tested.

In FIG. 12B shows representative graphs demonstrating the efficacy of anti-αvβ8 antibody (ADWA11) at 1, 3, 10 and 20 mg/kg as monotherapy and isotype control (mIgG1_4mut isotype (2B8)) in the EMT6 model of breast cancer in the top panel. The combination of anti-αvβ8 antibody ADWA11 at 1, 3, 10 and 20 mg/kg with anti-PD1 antibody (RMP1-14, 10 mg/kg) and rat IgG2a isotype (2A3) in the EMT6 model of breast cancer is also shown. Mice are treated with antibodies on days 0, 4 and 8 of the study, and tumor growth is measured with digital calipers three times a week and shown as tumor volume (length x width x width x 0.5).

In FIG. 13 shows representative graphs demonstrating the effectiveness of combination treatment of anti-αvβ8 (ADWA11), anti-41BB (MAB9371), anti-CTL4 (9D9), mIgG1_4mut isotype (2B8) and rat IgG2a isotype (2A3) in the EMT6 model of breast cancer. Tumor growth is measured with digital calipers three times a week and shown as tumor volume (length x width x width x 0.5), the mean tumor volume +/- SEM in each treatment group is plotted until there are less than 8 tumors remaining in each group /10 mice, and survival is defined as the time to reach 1000 mm ³ . The combination of anti-4-1BB and ADWA11 or anti-CLTA4 and ADWA11 treatment inhibited tumor growth and improved overall survival to a greater extent than other combinations tested.

In FIG. 14A shows representative graphs demonstrating that the combination of anti-αvβ8 antibody and radiation therapy (ADWA11+5Gy radiation group) inhibits tumor growth and improves overall survival to a greater extent than isotype control radiation therapy (mIgG4mut (2B8) + 5Gy radiation group) in the CT26 tumor model. Tumor growth is measured with digital calipers three times a week and shown as tumor volume (length x width x width x 0.5), the mean tumor volume +/- SEM in each treatment group is plotted until there are less than 8 tumors remaining in each group /10 mice, and survival is defined as the time to reach 1000 mm ³ . *p <0.05 k without treatment, **P <0.05 k radiation+2B8.

In FIG. 14B top graph shows the density of cells expressing CD45, CD8 and Granzyme B in CT26 tumor tissue collected on day 12 from mice treated on day 0, day 4 and day 8 with 10 mg/kg isotype control (Control) or ADWA11 antibody ( Anti-ITGαVβ8), n=6; p=P-value. The bottom graph shows CD45, CD8, GranzymeB, and IFNγ gene expression in tumor tissue collected 12 days after the first 10 mg/kg dose of isotype control (Isothia), ADWA11 antibody (Anti-ITGαVβ8), Isotype in combination with 5 Gy tumor-targeted radiation or ADWA11 antibody in combination with 5 Gy of tumor-targeted radiation. Antibody treatment is given intravenously on day 1, day 4 and day 8 of the study, and radiation therapy is given on day 5 of the study. Five mice are included in each treatment group; The mean and standard error are plotted.

In FIG. 15 shows representative graphs showing IHC analysis of the density of CD45 (total lymphocytes and myeloid cells), CD3 (total T cells), CD4 T cells, CD8 T cells, and Granzyme B (activated CD8 and NK cells) in the EMT6 tumor model. Treatment with ADWA11(2.4) (also referred to as ADWA11VH05-2/VK01(2.4) herein) increased the density of all cell types analyzed. N=10 per group, p value for two-tailed t-test is indicated in the graph.

In FIG. 16A is a diagram showing the treatment regimen for the CCK168 tumor model. Timelines for tumor cell implantation and intraperitoneal (i.p.) antibody injection are presented for the four treatment groups.

In FIG. 16B shows representative Kaplan-Meier survival curves using a tumor volume of 2000 mm ³ as the cutoff value for survival in the CCK168 tumor model. n=10 in each group. **p < 0.01 by Cox-Mantel log-rank test.

In FIG. 16C shows representative graphs showing individual growth curves of the tumors shown in FIG. 16B. Mice are sacrificed before the 45-day endpoint if tumors reach ≥2000 mm ³ or if extensive tumor ulceration is observed. Combination treatment with anti-PD1 antibody and ADWA11 synergistically inhibits tumor growth and improves overall survival to a greater extent than treatment with anti-PD1 antibody, ADWA11 or isotype control alone, or their individual effects simply added together. Data shown are representative of 3 independent biological replicates.

In FIG. 17A shows a gating strategy for identifying tumor-infiltrating monocytes, macrophages, and dendritic cells. Live single cells are first gated with a reset gate including Ly6G, SiglecF, CD90, and B220 to exclude neutrophils, eosinophils, lymphocytes, and B cells. Negatively stained cells are then analyzed by flow cytometry and gated. Macrophages are identified as CD45+CD11b+CD64 ^high F4/80 ^high , and dendritic cells are identified as CD45+CD11b+F4/80-CD64-MHCII ^high CD11c ^high . From the macrophage population, profiles corresponding to immunostimulatory macrophages are identified as Ly6C ^high CD206 ^low , and immunosuppressive macrophages are identified as Ly6C ^low CD206 ^high . Reset channel Ly6G+SiglecF+CD90.2+B220+.

In FIG. 17B is a representative graph showing cell surface staining for αvβ8 integrin analyzed as part of a multicolor flow cytometry panel in the disaggregated tumors described in FIG. 17A. Representative flow cytometry plots show fluorescence minus one control staining (FMO) and ADWA11 antibody staining in the CCK168 tumor model. N=4 tumors.

In FIG. 18A shows representative graphs showing ADWA11 staining on CD45 negative cells isolated from CCK168. Live single cells are analyzed for the presence of CD45. 4% of CD45 negative cells in the CCK168 tumor model were positive for αvβ8 expression using the ADWA11 antibody.

In FIG. 18B shows αvβ8 expression in CCK168, CT26 and EMT6 cell lines. Live single cells were analyzed for αvβ8 expression by flow cytometry using isotype and anti-αvβ8 (ALDWA11) staining antibodies. CCK168 and EMT6 cell lines had detectable expression of αvβ8, while CT26 cells did not.

In FIG. 19A shows representative graphs showing total intratumoral CD8+ T cell counts in mice bearing CCK168 tumors treated with control antibodies alone, anti-PD1, ADWA11, or a combination of anti-PD1 and ADWA11. Representative flow cytometry plots for CD4 and CD8 expression by all CD45+ cells are shown along with the ratio of CD8+ cells to CD45+ cells for each mouse in each group.

In FIG. 19B shows representative immunofluorescence micrographs of CCK168 tumor sections collected from mice in each treatment group, stained for anti-CD8 and DAPI. Scale bar: 50 µm.

In FIG. 19C shows representative flow cytometry plots showing intracellular staining for Granzyme B in cells gated for CD8 expression, along with the proportion of CD8+ cells expressing detectable Granzyme B in each mouse in each group.

In FIG. 19D shows representative flow cytometry plots showing immunostimulatory macrophages (Ly6c ^high , CD206 ^low ) and immunosuppressive macrophages (CD206 ^high , Ly6c ^low ) for cells gated as CD45+Ly6G-CD11b+CD64 ^high F4/80 ^high . From the macrophage population, immunostimulatory macrophages were identified as Ly6C ^high CD206 ^low and immunosuppressive macrophages were identified as Ly6C ^low CD206 ^high , along with the proportion of immunostimulatory macrophages for each mouse. Data in graphs are mean±SEM, n=10 per group. *p <0.05, **p <0.01, ***p <0.001 by one-way ANOVA.

In FIG. 20 shows representative graphs demonstrating ADWA11 staining on CD4+ and CD8+ T cells in the CCK168 tumor microenvironment. Live single CD45+ cells were gated for CD4 and CD8 and stained with ADWA11. Representative graphs are shown for mice from each of the 4 treatment groups.

In FIG. 21A shows representative graphs demonstrating immune elimination of CD8+ T cells. Micrographs show anti-CD8 immunostaining counterstained with DAPI in CCK168 tumors isolated after combination anti-PD-1/ADWA11 therapy with or without prior treatment with anti-CD8 eliminating antibody or isotype control antibody.

In FIG. 21B shows representative graphs showing average tumor growth curves for CCK168 tumors pretreated with anti-CD8 depleting antibody or isotype control antibody 24 hours prior to ADWA11/anti-PD-1 combination therapy.

In FIG. 21C shows representative graphs demonstrating the survival of mice bearing CCK168 tumors following ADWA11/anti-PD-1 combination therapy, pretreated one day earlier with either an anti-CD8 eliminating antibody or an isotype control antibody. Data are presented as proportion of survival, n=10 in each group. **p < 0.01 by Cox-Mantel log-rank test.

In FIG. 22A shows representative graphs showing CCK168 tumors collected from mice treated with either ADWA-11 or control antibody and stained with anti-CD8, F4/80 for macrophages, and phospho-SMAD3 for TGFβ (pS3) signaling. Low-power merged images and images showing only pSMAD3 are shown on the left, and high-magnification merged images and images for each individual antibody from the boxed areas are shown on the right.

In FIG. 22B shows representative graphs demonstrating quantitative analysis of pSmad3 (pS3) staining density in control and ADWA11 treated mice. ADWA11 treatment reduces pSmad3 density in CCK168 tumors.

In FIG. 23A shows representative plots with data extracted from The Cancer Genome Atlas (TCGA) for integrin-β8 mRNA expression in 30 different types of human cancer. Each dot represents an individual tumor sample. The results shown in this drawing are based on data collected by the TCGA Research Network.

In FIG. 23B shows representative graphs showing flow cytometry data of disaggregated cells from fresh, de-identified ovarian and renal cell carcinomas gated for mature monocytes (CD16+ monocytes), tumor-associated macrophages (CD14+ macrophages), two monocyte-derived dendritic cell populations ( BCDA1+ moDCs and BCDA3+ moDCs) or eosinophils, and stained for αvβ8 expression. Tissue from normal tonsils was also analyzed as a control. The gating strategy is shown in FIG. 23C; n=2 for each tumor type.

In FIG. 23C shows representative graphs demonstrating the gating strategy for human tumor biopsy samples. Live single CD45+ cells are gated for SSC-A(hi) to remove granulocytes. SSC-A(lo) was gated for HLA-DR+CD3- and stained for CD14 and CD16. CD14+CD16+ are referred to as CD16+ monocytes. CD16 cells are further stained for CD11c. CD14+CD11c+ cells are further stained with CD1c and CD141. CD1c+CD141- is designated as CD1c+ MoDC. CD141+CD1c- is designated as CD141+ MoDC. CD1c-CD141- cells are further stained with CD64 and the CD64+ population is designated CD14+TAMs.

In FIG. 24A is a representative graph showing the results of TMLC cell co-culture bioassays performed with ADWA11 concentrations ranging from 0.01 to 10 mg/ml. TGFβ activity is shown in relative luciferase units based on PAI-1 luciferase reporter activity. n=3 per dose of ADWA11, repeat 3 times.

In FIG. 24B is a representative graph showing the results of cell adhesion assays performed on plates coated with TGFβ1 latency associated peptide (LAP) in the presence of ADWA-11 at concentrations ranging from 0.001 to 10 mg/ml. Adherent cells are stained with crystal violet, and adhesion is expressed as absorbance at 595 nm. n=3 per dose of ADWA11, repeat 3 times.

In FIG. 24C shows representative graphs demonstrating that antibodies to integrin αvβ3, αvβ5, αvβ6, and αvβ8 are used for flow cytometry of wild-type, SW-480 colon carcinoma cells that do not express any of these integrins, or SW-480 cells transfected to express β3 (SW-itgb3), β6 (SW-itgb6) or β8 (SW-itgb8). Representative flow cytometry plots are shown for each antibody and cell type tested.

In FIG. 25A is a representative graph showing a two-compartment nonlinear pharmacokinetic model fitted to 0.1, 0.3 and 3 mg/kg i.v. dosing of ADWA11(2.4) in TG32 mice. Circles: received data. Lines: model adaptation.

In FIG. 25B is a representative graph showing a two-compartment pharmacokinetic model adjusted to 3 mg/kg i.v. dosage of ADWA11 2.4 in TG32 mice. Circles: received data. Lines: model adaptation.

In FIG. 26 shows representative graphs demonstrating the dual-compartment pharmacokinetics of ADWA11 2.4 in cynomolgus monkeys following single IV bolus doses of 4, 40 and 100 mg/kg, respectively. Circles: received data. Lines: model adaptation.

In FIG. 27A-27B are representative graphs illustrating the predicted human pharmacokinetics of ADWA11 2.4 following 12 mg/kg Q28D and 7 mg/kg Q14D. CP is the predicted plasma concentration; CAVG is the C avg or mean plasma concentration; NOAEL in rodents is C _ave with no observable level of adverse effects in rodents; and NHP NOAEL is C _ave without observable levels of side effects in non-human primates.

In FIG. 28 shows representative graphs showing Kaplan-Meier survival curves using a tumor volume of 2000 mm ³ as the cutoff value for survival for the CCK168 tumor model. Treatment groups include: isotype control, anti-PD1 antibody, ADWA11_4mut, and a combination of anti-PD1 antibody and ADWA11_4mut. Treatment with a combination of anti-PD1 antibody and ADWA11_4mut synergistically improved overall survival to a greater extent than treatment with anti-PD1 antibody, ADWA11_4mut or isotype control alone or the expected additive effect of combination treatments.

In FIG. 29A shows graphs depicting representative survival curves and FIG. 29B shows representative individual tumor growth curves of mice implanted subcutaneously with CT26 cells and treated with isotype control antibodies, anti-PD1, ADWA11_4mut, or a combination of anti-PD1 and ADWA11_4mut, plus 5 Gy of radiation on day 5. One group of mice treated with isotype control antibody did not receive radiation therapy. Data are presented as proportion of survival, n=10 in each group. ***p <0.001, ****p <0.0001 by Cox-Mantel log-rank test.

In FIG. 29C shows graphs depicting representative drug rechallenge tumors in CT26-treated mice that survived 50 days after initiation of treatment. Parental CT26 cells were implanted into the flank opposite the original tumor implantation site of CT-26-treated mice 51 days after initiation of immunotherapy in combination with radiation therapy. Control mice do not receive radiation and have not previously been infected with tumor cells. Repeatedly administered mice are monitored for 30 days. Control, n=10; RT plus anti-PD1, n=3; RT plus ADWA11_mut, n=5; RT plus ADWA11_mut and anti-PD1, n=7.

In FIG. 30A shows graphs depicting representative survival curves and FIG. 30B shows graphs depicting representative individual growth curves in mice orthotopically implanted with EMT6 cells following treatment with an isotype control antibody, ADWA11_4mut, 4-1BB, anti-CTLA4, anti-PD-1, or a combination of ADWA-11_4mut and 4-1BB, anti-CTLA4 or anti-PD-1. Data are presented as survival rate, n=10 in each group. ****p <0.0001 by Cox-Mantel log-rank test

In FIG. 30C shows graphs depicting representative survival curves and FIG. 30D shows graphs depicting representative individual growth curves of mice treated with anti-CTLA4 or 4-1BB activator.

In FIG. 30E shows representative graphs demonstrating tumor rechallenge results in mice that survived 50 days with complete tumor regression after treatment with the synergistic combination of ADWA-11_4mut and anti-CTLA4 or the synergistic combination of ADWA-11_4mut and activator 4-1BB. Control mice had not previously been infected with tumor cells. Mice with repeated administration of the drug are assessed for 30 days. n=5 control, n=6 ADWA-11_4mut+anti-CTLA4, n=5 ADWA-11_4mut+4-1BB.

In FIG. 31A depicts analysis of gene mRNA expression in MC38 tumor tissue using CD8a and GranzymeB specific taqman probes. Tumor tissue is collected 12 days after the first dosing of isotype control or ADWA11 2.4 antibody at the indicated doses. Treatment was administered intravenously on day 1, day 5, and day 9 of the study, with 5 mice included in each treatment group. * = p-value <0.05.

In FIG. 31B shows representative graphs demonstrating tumor growth rate in the MC38 tumor model in mice treated with isotype (10 mg/kg), ADWA11 2.4 (10 mg/kg), anti-PD-1 antibody (RMP1-14, 10 mg/kg) and a combination of ADWA11 2.4 (10 mg/kg) and anti-PD1 antibody (RMP1-14, 10 mg/kg). Additionally, representative graphs showing tumor growth rates of MC38 tumors in mice treated with ADWA11 (anti-ITGAVB8 (ADWA11_mIgG_4mut) antibody at 0.03, 0.3, 3 and 30 mg/kg in combination with 10 mg/kg anti-PD -1 antibody (RMP1-14) For all schedules, antibodies were administered on days 1, 5 and 9 of the study.

DETAILED DESCRIPTION

Disclosed herein are antibodies and an antigen binding fragment thereof that specifically bind to αvβ8 integrin (e.g., human αvβ8 integrin) and additionally, antibodies that antagonize αvβ8 integrin activity (e.g., antagonize TGFβ activation, antagonize mediation of TGFβ production, antagonize modulation of Tregs and Th17 cells) or their interaction with TGFβ1 and TGFβ3 or the release of active TGFβ. Also presented are methods for producing anti-αvβ8 integrin antibodies, compositions containing anti-αvβ8 integrin antibodies, and methods for using anti-αvβ8 integrin antibodies. In some embodiments, recombinant, for example humanized, antibodies that bind αvβ8 integrin (eg, human αvβ8 integrin) are provided. In some embodiments, also provided are humanized heavy chain and light chain antibodies that are capable of generating antibodies that bind αvβ8 integrin. In some embodiments, humanized antibodies are provided, heavy chains and light chains containing one or more specific complementarity determining regions (CDRs). In some embodiments, humanized anti-αvβ8 integrin antibodies have altered effector functions. In some embodiments, antibodies of the invention have reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) relative to otherwise identical anti-αvβ8 integrin antibodies of the invention.

Provided are polynucleotides encoding antibodies that bind αvβ8 integrin (eg, human αvβ8 integrin) or an antigen-binding fragment thereof. Polynucleotides encoding antibody heavy chains or light chains are also provided. Provided are host cells that express anti-αvβ8 integrin antibodies, including humanized antibodies. Treatment methods using anti-αvβ8 integrin antibodies, including humanized antibodies, are presented.

Anti-αvβ8 integrin antibodies and their antigen binding fragment, including humanized antibodies, can be used to prevent, treat and/or alleviate diseases, disorders or conditions caused by and/or associated with aberrant (eg, elevated) TGFβ signaling. Such diseases, disorders or conditions include cancer (eg, control of proliferation of cancer cells with aberrant (eg, increased) TGFβ signaling).

Without wishing to be bound by any particular theory, mature TGFβ is present in an inactive or latent form in complex with a latency associated peptide (LAP) domain. Binding of αvβ8 integrin to LAP releases active TGFβ (eg, TGFβ1 and TGFβ3). Reduced αvβ8 integrin binding to LAP may prevent the release of active TGFβ, thereby reducing TGFβ signaling. TGFβ is known to have immune suppressive effects, for example, in the tumor microenvironment, and therefore reduction of TGFβ activity and/or antibody signaling described herein may cause activation of an immune response, such as an antitumor response in vivo . Thus, the antibodies and antigen-binding fragment thereof herein enable selective antagonism of TGFβ activity in the immune system and/or tumor microenvironment, thereby improving the antitumor immune response in the subject. In some embodiments, the antibodies disclosed herein and their antigen binding fragment against αvβ8 integrin have been shown to cause tumor growth suppression and/or complete tumor regression in animal models of certain cancers, including squamous cell carcinoma, breast cancer, and colon cancer. , alone or in combination with other immunomodulators, such as checkpoint inhibitor modulators (eg, PD-1, PD-L1, CTLA-4 inhibitors, or 4-1BB agonists) or anticancer therapies, such as radiotherapy. Thus, anti-αvβ8 integrin antibodies and their antigen binding fragment, including humanized antibodies, can be used, alone or in combination with a second therapy, for the prevention, treatment and/or amelioration of cancer, for example, a solid tumor, for example, a solid tumor selected from : renal cell carcinoma (RCC), ovarian cancer, or squamous cell carcinoma of the head and neck (SCCHN).

Section headings used in this document are for organizational purposes only and should not be construed as a limitation of the subject matter described.

All references cited herein, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference as if each individual reference were specifically identified for inclusion by reference in its entirety.

The methods and procedures described or referred to herein are generally well understood and are commonly used by those skilled in the art using traditional methodology, such as, for example, the widely used methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3 ^rd . edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993); and their updated versions.

I. DEFINITIONS

The present invention can be better understood by referring to the following detailed description of exemplary embodiments of the present invention and the examples included therein.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. In the event of any conflict, this specification, including definitions, governs.

In addition, unless the context otherwise requires or is expressly indicated, terms in the singular shall include the plural and terms in the plural shall include the singular.

Aspects and embodiments herein are intended to include “consisting of” and/or “consisting essentially of” aspects and embodiments. As used herein, the singular forms "a", "an" and "the" include plural references unless otherwise indicated.

In this application, the use of “or” means “and/or” unless explicitly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of “or” refers to more than one preceding independent or dependent claim.

"About" or "approximately", when used in connection with a measurable numerical variable, refers to the specified value of the variable and to all values of the variable that are within the experimental error of the specified value (for example, within the 95% confidence interval for the mean) or within within 10 percent of the specified value, whichever is greater. Numeric ranges include numbers that define a range.

Although the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values set forth in the specific examples are stated as accurately as possible. However, any numerical value by its nature contains certain errors, necessarily resulting from the standard deviation found in the corresponding test measurements. In addition, it should be understood that all ranges described herein include any and all sub-ranges contained therein. For example, the stated range "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and ending with a maximum value of 10 or less, such as 5.5 to 10.

In this specification and claims, the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers. Unless the context otherwise requires, terms in the singular shall include the plural and terms in the plural shall include the singular. Any example(s) following the term “for example” or “for example” are not intended to be exhaustive or limiting.

It is understood that wherever embodiments are described herein by the term “comprising”, similar embodiments described by the terms “consisting of” and/or “consisting essentially of” are also provided.

If aspects or embodiments of the invention are described in terms of a Markush group or other group of alternatives, the present invention covers not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, but also the main group is missing one or several group members. The present invention also provides for the express exclusion of one or more group members in the claimed invention.

It should be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting. In this description and in the following claims, reference will be made to a number of terms which are intended to have the following meanings.

The term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide or antibody or fragment thereof) represents a molecule that, by virtue of its origin or source of production (1) is not related to the naturally associated components that accompany it in its native state, (2) contains essentially no other molecules of the same species, (3) is expressed by a cell of a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system other than the cell from which it naturally originates will be "isolated" from its naturally associated components. The molecule may also be found to be substantially free of naturally associated components when isolated using purification methods well known in the art. The purity or homogeneity of a molecule can be determined by a number of methods well known in the art. For example, the purity of a polypeptide sample can be analyzed by polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be achieved by HPLC or other purification means well known in the art.

As used herein, "substantially pure" means that the species of the item is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition) and, preferably, the substantially purified fraction is the composition wherein the species of the entity (eg, a glycoprotein, including an antibody or receptor) constitute at least about 50 percent (on a molar basis) of all macromolecular species present. Typically, the substantially pure composition will comprise greater than about 80 percent of the total macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the species of the object are purified to be substantially homogeneous (impurities cannot be detected in the composition by conventional detection methods), where the composition consists of essentially one macromolecular species. In certain embodiments, the substantially pure material is at least 50% pure (e.g., free of impurities), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98 % pure and most preferably at least 99% pure.

The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence comparison. In the art, “identity” also means the degree of sequence relatedness between a polypeptide or nucleic acid sequence molecule, as the case may be, as measured by compatibility between strands of nucleotide or amino acid sequences. "Identity" measures the percentage of identical matches between two or more sequences with the gap alignment considered by a particular mathematical model of computer programs (i.e., "algorithms").

The term "similarity" is a related concept but, unlike "identity", refers to a measure of similarity that includes both identical matches and conservative matches. Since conservative substitutions apply to polypeptides but not to nucleic acid molecules, similarity applies only to polypeptide sequence comparisons. If two polypeptide sequences have, for example, 10 of 20 identical amino acids and all the rest are non-conservative substitutions, both the percentage identity and similarity will be 50%. If in the same example there are 5 more positions that have conservative substitutions, then the percent identity will remain 50%, but the percent similarity will be 75% (15 out of 20). Therefore, in cases where conservative substitutions exist, the degree of similarity between two polypeptide sequences will be higher than the percentage of identity between the two sequences.

"Fragments" or "parts" of a polypeptide or antibody in accordance with the invention can be obtained by truncation, for example, by removing one or more amino acids from the N and/or C termini of the polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids can be removed from the N and/or C terminus in this manner. Fragments can also be created by one or more internal deletions.

An antibody variant may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions and/or insertions from the specific sequences and fragments discussed above. “Deletion” variants may comprise deletion of individual amino acids, deletion of small groups of amino acids, such as 2, 3, 4 or 5 amino acids, or deletion of large amino acid regions, such as deletion of specific amino acid domains or other features. "Insertion" variants may comprise the insertion of individual amino acids, the insertion of small groups of amino acids, such as 2, 3, 4 or 5 amino acids, or the insertion of large amino acid regions, such as the insertion of specific amino acid domains, or other features. "Substitution" options preferably involve replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be replaced by an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. .

Substitution variants have at least one amino acid residue removed from the antibody molecule and another residue inserted in its place. The most interesting sites for replacement mutagenesis include hypervariable regions, but changes in framework regions are also considered. Conservative substitutions are shown below under the heading “conservative substitutions.” If such substitutions result in a change in biological activity, then more significant changes, referred to as "exemplary substitutions" shown below or further described below with reference to amino acid classes, can be introduced and the products screened.

Amino acids and substitutions

Original balance Conservative substitutions Typical replacements Alanine Ala (A) Val Val; Leu; Ile arginine Arg(R) Lys Lys; Gln; Asn asparagine Asn(N) Gln Gln; His; Asp, Lys; Arg aspartic acid Asp (D) Glu Glu; Asn cysteine Cys(C) Ser Ser; Ala glutamine Gln (Q) Asn Asn; Glu glutamic acid Glu(E) Asp Asp; Gln glycine Gly (G) Ala Ala histidine His (H) Arg Asn; Gln; Lys; Arg Isoleucine Ile(I) Leu Leu; Val; Met; Ala; Ph; Norleucine leucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe lysine Lys (K) Arg Arg; Gln; Asn methionine Met (M) Leu Leu; Ph; Ile Phenylalanine Phe (F) Tyr Leu; Val; Ile; Ala; Tyr proline Pro (P) Ala Ala serine Ser(S) Thr Thr threonine Thr (T) Ser Ser tryptophan Trp (W) Tyr Tyr; Phe tyrosine Tyr(Y) Phe Trp; Ph; Thr; Ser valine Val(V) Leu Ile; Leu; Met; Ph; Ala; Norleucine

Significant modifications of the biological properties of an antibody are achieved by selecting substitutions that vary significantly in their effect on maintaining (a) the structure of the polypeptide backbone at the site of the substitution, for example, a beta-pleated or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the main part of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

i. non-polar: Norleucine, Met, Ala, Val, Leu, Ile;

ii. polar without charge: Cys, Ser, Thr, Asn, Gln;

iii. acidic (negatively charged): Asp, Glu;

iv. basic (positively charged): Lys, Arg;

v. residues that affect chain orientation: Gly, Pro; And

vi. aromatic: Trp, Tyr, Phe, His.

Non-conservative replacements are made by replacing a member of one of these classes with another class.

One type of substitution, for example, that can be made is to replace one or more cysteines in the antibody, which may be chemically reactive, with another residue, such as, but not limited to, alanine or serine. For example, a non-canonical (eg, non-preferred or common) cysteine may be substituted. The substitution may be in the CDR or framework region of the variable domain or in the constant region of the antibody. In some embodiments, the cysteine is canonical (eg, preferred or most abundant). Any cysteine residue not involved in maintaining proper antibody conformation can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine linkage(s) may be added to an antibody to increase its stability, especially if the antibody is an antibody fragment such as an Fv fragment.

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, fat, polypeptide, etc. through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term covers not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding fragment thereof that competes with the intact antibody for specific binding, fusion proteins containing an antigen binding fragment, and any other modified configurations immunoglobulin molecules that include an antigen recognition site. Antigen binding fragments include, for example, Fab, Fab', F(ab') ₂ , Fd, Fv, antibody domain (dAb, e.g. shark and camel antibodies), fragments including complementarity determining regions (CDRs), single chain antibody variable fragment ( scFv), maxibodies, minibodies, diabodies, tribodies, tetrabodies, v-NAR and bis-scFv and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigenic binding to the polypeptide.

An antibody includes any class of antibody such as IgG, IgA, or IgM (or subclasses thereof), and the antibody does not necessarily belong to any particular class. Depending on the amino acid sequences of the heavy chain (HC) constant region of an antibody, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant regions that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), used interchangeably herein, refer to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, αvβ8 integrin). It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen binding fragment” of an antibody include (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of one arm of the antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) dedicated complementarity determining region (CDR), disulfide-linked Fv (dsFv) and anti-idiotypic (anti-Id) antibodies and intrabodies. In addition, although the two domains of the Fv fragment, VL and VH, are encoded for separate genes, they can be combined using recombinant methods, a synthetic linker that allows them to be obtained as separate protein chains in which the VL and VH regions are paired to produce monovalent molecules (known as single chain Fv (scFv)); see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain antibodies are also encompassed by the term "antigen binding fragment" of an antibody. Other forms of single chain antibody, such as diabodies, are also covered. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby causing the domains to pair with complementary domains of the other chain and create two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

Antibodies can come from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc. or other animals such as birds (eg chickens), fish (eg sharks) and camels (eg llamas).

An antibody "variable region" refers to an antibody light chain variable region (VL) or an antibody heavy chain variable region (VH), either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) connected by three "complementarity determining regions (CDRs)", also known as hypervariable regions (HVRs), and are involved in the formation of antigen-binding antibody site. If variants of such variable regions are desired, particularly with substitution at amino acid residues outside the CDR region (i.e., in the framework region), a suitable amino acid substitution, preferably a conservative amino acid substitution, can be identified by comparing said variable regions with the variable regions of other antibodies. , which contain CDR1 and CDR2 sequences in the same canonical class as the specified variable region (Chothia and Lesk, J. Mol. Biol. 196(4): 901-917, 1987).

In certain embodiments, the final determination of the CDR and identification of the residues constituting the antibody binding site is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, this may be accomplished by any of a variety of techniques known to those skilled in the art, such as x-ray crystallography. In certain embodiments, various analysis techniques may be used to identify or approximate CDR regions. In certain embodiments, various analysis techniques may be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, Kabat determination, Chotia determination, AbM determination, contact determination, and conformational determination.

There are several methods in the art for numbering the amino acid residues that form a CDR. The Kabat numbering method is the standard for numbering residues in an antibody, and is also commonly used to identify CDRs. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chotia definition is similar to the Kabat definition, but the Chotia definition takes into account the positions of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. AbM determination uses an integrated set of computer programs produced by the Oxford Molecular Group that model the structure of the antibody. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. Definitions AbM models the tertiary structure of an antibody from a primary sequence using a combination of knowledge bases and ab initio methods such as those described in Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl. 3:194–198.

Contact determination is based on analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to here as “conformational determination” of CDRs, CDR positions can be identified as residues that make an enthalpic contribution to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Other definitions of CDR boundaries may not strictly follow one of the approaches presented above, but nevertheless overlap with at least part of the CDR according to Kabat, although they may be shortened or expanded depending on predictive or experimental discoveries that certain residues or groups of residues have little effect on antigen binding. As used herein, CDR can refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may apply CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the Cabot, Chotia, extended, AbM, contact, and/or conformational definitions.

"Contact residue" as used herein in relation to an antibody or antigen specifically bound thereto, refers to an amino acid residue present in the antibody/antigen containing at least one heavy atom (i.e., non-hydrogen) that is present in within 4 Å or less of the heavy atom of an amino acid residue present on the related antibody/antigen.

"Framework" (FR) residues are residues of the antibody variable domain other than the CDR residues. The VH or VL domain framework regions contain four framework sub-regions, FR1, FR2, FR3 and FR4, interspersed with CDRs in the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Residues in the variable domain are typically numbered according to Kabat, which provides the numbering system used for heavy chain variable domains or light chain variable domains in antibody formulation. See Kabat et al., 1991, Sequences of Proteins of Immunological Interest, ^5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. By using this numbering system, an actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the abbreviation or insertion in the FR or CDR of the variable domain. For example, a heavy chain variable domain may include one amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (eg, residues 82a, 82b and 82c according to Kabat) after residue 82 of the heavy chain FR. Kabat residue numbering can be determined for a given antibody by aligning regions of homology between the antibody sequence and the “standard” Kabat numbering sequence. Different algorithms are available for assigning numbering according to Kabat. The algorithm implemented in version 2.3.3 of the Abysis release (www.abysis.org) can be used to assign Kabat numbering to the variable regions CDR-L1, CDR-L2, CDR-L3, CDR-H2 and CDR-H3, and then define the AbM can be applied to CDR-H1.

As used herein, "monoclonal antibody" refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. individual antibodies making up a population that are identical except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific because they are directed against a single antigenic site. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. The modifier “monoclonal” indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method first described in Kohler and Milstein, 1975, Nature 256:495, or can be produced by recombinant DNA methods described in US Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. As used herein, a “humanized” antibody refers to forms of non-human (eg, murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding antibody sub-sequences) that contain minimal sequence derived from non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient CDR are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. A humanized antibody may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences, but are included to further improve or optimize the effectiveness of the antibody.

The antibody or antigen-binding fragment thereof of the invention may be affinity mature. For example, affinity mature antibody can be produced by methods known in the art (Marks et al., 1992, Bio/Technology, 10:9-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91 :3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and WO2004/058184).

A “human antibody” is one that has an amino acid sequence that matches that of an antibody produced by a human and/or produced using any of the methods for producing a human antibody as described herein. This definition of human antibody specifically excludes a humanized antibody containing non-human antigen binding residues.

The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. , or vice versa. The term also includes an antibody comprising a V region from one individual from one species (eg, a first mouse) and a constant region from another individual from the same species (eg, a second mouse).

The term “antigen (Ag)” refers to a molecular substance used to immunize an immunocompetent vertebrate to produce an antibody (Ab) that recognizes an Ag, or to screen an expression library (eg, a phage, yeast, or ribosome display library, among others). Here, Ag is referred to more broadly and typically includes target molecules that are specifically recognized by Ab, thereby including fragments or mimetics of the molecule used in immunization to raise Ab, or in library screening to select Ab. Thus, for antibodies of the invention that bind to αvβ8 integrin, full-length αvβ8 integrin from mammalian species (e.g., human, monkey, mouse and rat αvβ8 integrin), including monomers and multimers, such as its dimers, trimers, etc., as well as truncated and other variants of αvβ8 integrin, are called antigen.

In general, the term "epitope" refers to the region or region of an antigen (eg, protein, nucleic acid, carbohydrate or fat, etc.) to which an antibody specifically binds, i.e. zone or area in physical contact with the antibody. Thus, the term "epitope" refers to that part of the molecule that is capable of being recognized and bound by an antibody at one or more antigen-binding regions of the antibody. Typically, an epitope is defined in the context of a molecular interaction between an “antibody or antigen-binding portion thereof” (Ab) and its corresponding antigen. Epitopes often consist of a surface grouping of molecules, such as amino acids or sugar side chains, and have certain three-dimensional structural characteristics as well as certain charge characteristics. In some embodiments, the epitope may be an epitope of a protein. Protein epitopes can be linear or conformational. In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" contains non-contiguous polypeptides (or amino acids) in an antigenic protein to which an epitope-specific antibody binds. The term "antigenic epitope", as used herein, is defined as that portion of an antigen to which an antibody can specifically bind, as determined by any method known in the art, such as conventional immunoassays. Alternatively, during the discovery process, antibody generation and characterization can provide information on desired epitopes. From such information it is then possible to competitively screen antibodies for binding to the same epitope. The approach to this involves performing competition and cross-competition studies to look for antibodies that compete and cross-compete with each other to bind to the αvβ8 integrin, for example, antibodies compete to bind to an antigen.

An antibody that “preferentially binds” or “specifically binds” (used interchangeably herein) to an epitope is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule exhibits "specific binding" or "preferential binding" if it interacts or associates more frequently, more rapidly, for a longer duration, and/or with greater affinity with a particular cell or substance than with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily and/or for a longer duration than it binds to other substances. Also, an antibody “specifically binds” or “predominantly binds” to a target if it binds with greater affinity, avidity, more readily and/or for a longer duration to that target in a sample than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to an αvβ8 integrin epitope is an antibody that binds that epitope with greater affinity, avidity, more readily, and/or longer duration than it binds other αvβ8 integrin epitopes or non-αvβ8 integrin epitopes . It is also clear when reading this definition, for example, that an antibody (or fragment or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, a reference to linking means preferential linking. “Specific binding” or “preferential binding” includes a compound, such as a protein, nucleic acid, antibody, or the like, that recognizes and binds to a specific molecule, but does not recognize or bind substantially to other molecules in the sample. For example, an antibody or peptide receptor that recognizes and binds to a cognate ligand or binding partner (e.g., anti-αvβ8 integrin antibodies that bind αvβ8 integrin) in a sample, but does not substantially recognize or bind to other molecules in the sample, specifically binds to this cognate ligand or binding partner. Thus, under specified assay conditions, a particular binding moiety (e.g., an antibody or an antigen-binding fragment thereof or a receptor or ligand-binding moiety thereof) binds preferentially to a particular target molecule and does not bind in significant amounts to other components present in the test sample .

A variety of assay formats can be used to select an antibody or peptide that specifically binds a molecule of interest. For example, ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (FortéBio, Inc., Menlo Park, CA), and Western blotting are some of the many assays that can be used to identify an antibody that specifically interacts with an antigen, or an antigen-binding fragment thereof, or a receptor or ligand-binding portion thereof that specifically binds a related ligand or binding partner. Typically, a specific or selective response will be at least twice the background signal or noise, more often, more than 10 times the background, even more often, more than 50 times the background, more often, more than 100 times the background more than background, but even more frequently, more than 500 times the background, even more than often, more than 1000 times the background, and even more typically, more than 10,000 times the background. Additionally, an antibody “specifically binds” an antigen when the equilibrium dissociation constant (K _D ) is ≤1 μM, preferably ≤100 nM, more preferably ≤10 nM, even more preferably ≤100 pM, even more preferably ≤10 pM and even more preferably, ≤1 pM. In some embodiments, the antibody “specifically binds” an antigen when the equilibrium dissociation constant (K _D ) is ≤7 nM.

The term "binding affinity" is used here as a measure of the strength of the non-covalent interaction between two molecules, for example, an antibody or fragment thereof and an antigen. The term binding affinity is used to describe monovalent interactions (intrinsic activity).

Additionally, to determine the binding affinity of the anti-αvβ8 integrin antibody to cells expressing αvβ8 integrin, cell binding experiments can be performed to determine apparent affinity. The apparent binding affinity of an antibody to cells expressing the target can be calculated as the EC ₅₀ equilibrium binding titration curves in which the geometric mean fluorescence intensity (gMFI) of the antigen binding population is quantified by flow cytometry.

The binding affinity between two molecules, for example, an antibody or fragment thereof and an antigen, through a monovalent interaction, can be quantified by determining the dissociation constant (K _D ). In turn, K _D can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant k _a (or k _on ) and the dissociation rate constant k _d (or k _off ), respectively. K _D is related to k _a and k _d through the equation K _D = k _d / k _a . The value of the dissociation constant can be determined directly by well known methods, and can be computerized even for complex mixtures by methods such as those described, for example, by Caceci et al. (1984, Byte 9: 340-362). For example, K _D can be determined using a dual nitrocellulose filter binding assay such as disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for assessing the binding ability of ligands such as antibodies to target antigens are known in the art, including, for example, ELISA, Western blot, RIA and flow cytometry assays, and other assays presented throughout herein. Binding kinetics and binding affinity of an antibody can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example, using the Biacore™ or KinExA system.

A competitive binding assay can be performed in which the binding of an antibody to an antigen is compared with the binding of a target by another ligand of that target, such as another antibody or a soluble receptor that binds to the target in a different manner. The concentration at which 50% inhibition occurs is known as K _i . Under ideal conditions, K _i is equivalent to K _D . The value of K _i will never be less than K _D , so the measurement of K _i can conveniently be substituted to give an upper limit for K _D .

Following the definition of binding affinities presented above, those associated with different molecular interactions, for example, a comparison of the binding affinities of different antibodies for a given antigen can be made by comparing the K _D values for individual antibody/antigen complexes. The K _D value for antibodies or other binding partners can be determined using methods well known in the art. One method for determining K _D involves the use of surface plasmon resonance, typically using a biosensor system such as the Biacore® system.

Also, the specificity of an interaction can be assessed by determining and comparing the K _D value of an interaction of interest, such as a specific antibody-antigen interaction, with the K _D value of an interaction of no interest, such as a control antibody known to not bind αvβ8 integrin.

An antibody that specifically binds its target can bind its target with high affinity, that is, exhibiting a low _KD as discussed above, and can bind other, non-target molecules with lower affinity. For example, the antibody may bind to off-target molecules with a K _D of 1 x 10 ^-6 M or more, more preferably 1 x 10 ^-5 M or more, more preferably 1 x 10 ^-4 M or more, more preferably 1 x 10 ^-3 M or more, even more preferably 1 x 10 ^-2 M or more. The antibody of the invention is preferably capable of binding to its target with an affinity that is at least twice, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or 10,000-fold or greater than its affinity for binding to another molecule, non-αvβ8 integrin.

The term “compete” as used herein with respect to an antibody means that the first antibody or antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding fragment thereof such that the binding effect of the first antibody on its cognate epitope is markedly reduced in the presence of the second antibodies compared to the binding of the first antibody in the absence of the second antibody. There may, but is not necessarily, an alternative where the binding of the second antibody to its epitope is also markedly reduced in the presence of the first antibody. That is, the first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, if each antibody detectably inhibits the binding of another antibody to its cognate epitope or ligand to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). The present invention covers both competitive and cross-competitive antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of ordinary skill in the art will understand, based on the teachings presented herein, that such competitive and/or cross-competitive antibodies are included and can be used for the methods described herein.

Standard competition assays can be used to determine whether antibodies compete with each other. One suitable antibody competition assay involves the use of Biacore technology, which can measure the extent of interactions using surface plasmon resonance (SPR) technology, typically using a biosensor system (such as the BIACORE® system). For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay to measure antibody competition uses an ELISA-based approach.

In addition, a high-throughput method for “sorting” antibodies based on competition is described in international patent application No. WO2003/48731. Competition is present if one antibody (or fragment) reduces the binding of another antibody (or fragment) to αvβ8 integrin. For example, a competitive sequential binding assay with sequential addition of different antibodies may be used. The first antibody may be added to achieve binding that is close to saturation. The second antibody is then added. If the binding of the second antibody to the αvβ8 integrin is undetectable or significantly reduced (e.g., at least about 10% at least about 20% at least about 30% at least about 40% at least about 50% at least about 60% at least about 70% at least about 80% or at least about 90% reduction) compared to a parallel assay in the absence of the first antibody (the value for which can be set to 100%), the two antibodies are considered to compete with each other .

In addition, a generic antibody epitope binding assay using domain swapping between human and mouse αvβ8 integrin proteins to evaluate potential epitopes among multiple antibodies is presented in Example 9. One of ordinary skill in the art will appreciate, armed with the concepts presented herein, that a variety of assays exist assays known in the art that can be used to determine the target binding of at least two antibodies relative to each other, and such assays are included herein.

Anti-αvβ8 integrin antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope to which it binds, or “epitope mapping.” There are many methods in the art for mapping and characterizing the location of epitopes on proteins, including dissolution of the crystal structure of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. As a further example, epitope mapping can be used to determine the sequence to which an anti-αvβ8 integrin antibody binds. Epitope mapping is commercially available from various sources, such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, i.e. contained in a single strand of amino acids, or a conformational epitope formed by the three-dimensional interaction of amino acids that may not necessarily be contained in a single strand. Peptides of varying lengths (eg, at least 4-6 amino acids in length) can be isolated or synthesized (eg, recombinantly) and used for anti-αvβ8 integrin antibody binding assays.

In addition, the epitope to which an anti-αvβ8 integrin antibody binds can be determined in a systemic screen using overlapping peptides derived from the αvβ8 integrin sequence (eg, human αvβ8 integrin sequence) and determining antibody binding. According to gene fragment expression assays, the open reading frame encoding the αvβ8 integrin can be fragmented either randomly or by specific genetic constructs, and the reactivity of the expressed αvβ8 integrin fragments with the tested antibody can be determined. Gene fragments, for example, can be obtained by PCR and transcribed and translated into protein in vitro , in the presence of radioactive amino acids. Antibody binding to radiolabeled αvβ8 integrin fragments is then determined by immunoprecipitation and gel electrophoresis.

Specific epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in simple binding assays. In a further example, antigen mutagenesis, domain swap experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or essential for epitope binding. For example, alanine scanning mutagenesis experiments can be performed using mutant αvβ8 integrin in which various residues of the αvβ8 integrin polypeptide have been replaced with alanine. By assessing antibody binding to mutant αvβ8 integrin, the importance of specific αvβ8 integrin residues for antibody binding can be assessed.

Another method that can be used to characterize an anti-αvβ8 integrin antibody is the use of competition assays with other antibodies known to bind to the same antigen, i.e. different fragments on αvβ8 integrin, to determine whether the anti-αvβ8 integrin antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

In addition, the epitope for a given antibody/antigen binding pair can be determined and characterized to varying levels of detail using a variety of experimental and computerized epitope mapping techniques. Experimental methods include mutagenesis, x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen/deuterium exchange mass spectrometry (H/D-MS) and various competitive binding techniques well known in the art. Because each method is based on a unique principle, the description of an epitope is closely related to the way in which it was determined. Thus, the epitope for a given antibody/antigen pair will be determined differently depending on the epitope mapping method used.

At the most detailed level, the epitope for the interaction between Ag and Ab can be determined using spatial coordinates defining the atomic contacts present in the Ag-Ab interaction, as well as information about their relative contribution to the thermodynamics of binding. At a less detailed level, an epitope can be characterized using spatial coordinates defining the atomic contacts between Ag and Ab. At an even less detailed level, an epitope can be characterized by the amino acid residues it contains, as determined by a particular criterion, such as the distance between atoms (eg, heavy, ie, non-hydrogen atoms) in Ab and Ag. At an even less detailed level, an epitope can be characterized in terms of function, for example, through competitive binding to other Abs. An epitope can also be defined more generally as containing amino acid residues for which substitution with another amino acid would alter the characteristics of the interaction between Ab and Ag (eg, using alanine scanning).

From the fact that descriptions and definitions of epitopes, depending on the epitope mapping method used, are obtained at different levels of detail, it follows that comparisons of epitopes for different Abs on the same Ag can similarly be carried out at different levels of detail.

Epitopes described at the amino acid level, such as those determined by X-ray structure, are considered identical if they contain the same set of amino acid residues. Epitopes are said to overlap if the epitopes share at least one amino acid. Epitopes are considered separate (unique) if the epitopes do not share amino acid residues.

Competitive binding epitopes are said to overlap if the binding of the corresponding antibodies is mutually exclusive, i.e. binding of one antibody precludes simultaneous or sequential binding of another antibody. Epitopes are said to be distinct (unique) if the antigen is capable of binding both corresponding antibodies simultaneously.

The definition of the term "paratope" is derived from the above definition of "epitope" with a change in perspective. Thus, the term “paratope” refers to the region or region of an antibody that specifically binds an antigen, that is, the amino acid residues of the antibody that contact the antigen (αvβ8 integrin or portion thereof), as “contact” is defined elsewhere herein.

The epitope and paratope for a given antibody/antigen pair can be identified by conventional methods. For example, the overall location of an epitope can be determined by assessing the ability of an antibody to bind to various fragments or variants of αvβ8 integrin polypeptides. Certain amino acids within the αvβ8 integrin contact the antibody (epitope), and certain amino acids within the antibody that contact the αvβ8 integrin (paratope) can also be determined using standard methods such as those described in the examples. For example, the antibody and target molecule can be combined and the antibody/antigen complex can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the antibody and its target.

An antibody of the present invention may bind to the same αvβ8 integrin epitope or domain (eg, human αvβ8 integrin) as the antibodies of the invention that are specifically described herein. Assays and assays that can be used for the purpose of such identification include assays that assess competition for αvβ8 integrin binding between the antibody of interest and the αvβ8 integrin receptor in biological activity assays as described in Examples 1-26.

An antibody or antigen binding fragment thereof may have the ability to compete or cross-compete with another antibody of the invention for binding to αvβ8 integrin (eg, human αvβ8 integrin) as described herein. For example, an antibody of the invention may compete or cross-compete with antibodies described herein for binding to αvβ8 integrin or a suitable fragment or variant of αvβ8 integrin that is bound by antibodies described herein.

That is, if the first antibody competes with the second antibody to bind to the αvβ8 integrin, but does not compete if the second antibody binds to the αvβ8 integrin first, it is said to “compete” with the second antibody (also called unidirectional competition). When an antibody competes with another antibody, regardless of which antibody binds αvβ8 integrin first, then the antibody "cross-competes" for αvβ8 integrin binding with the other antibody. Such competitive or cross-competitive antibodies can be identified based on their ability to compete/cross-compete with a known antibody of the invention in standard binding assays. For example, SPR, such as using the Biacore™ system, ELISA assays or flow cytometry, can be used to demonstrate competition/cross-competition. This competition/cross-competition suggests that two antibodies bind to identical, overlapping, or similar epitopes.

Therefore, an antibody of the invention can be identified by a method involving a binding assay that assesses whether the test antibody is able to compete/cross-compete with a reference antibody for a binding site on a target molecule. Methods for performing competitive binding assays are described herein and/or are well known in the art. For example, they may involve binding a reference antibody of the invention to a target molecule using conditions under which the antibody can bind to the target molecule. The antibody/target complex can then be exposed to a test/second antibody and the extent to which the test antibody is able to displace the reference antibody of the invention from the antibody/target complexes can be assessed. An alternative method may involve contacting the test antibody with a target molecule under conditions that allow binding of the antibody, then adding a reference antibody of the invention that is capable of binding that target molecule, and assessing the extent to which the reference antibody of the invention is able to displace the test antibody from the complexes antibody/target or simultaneously contact the target (i.e., non-competitive antibodies).

The ability of a test antibody to inhibit binding of a reference antibody of the invention to a target demonstrates that the test antibody can compete with the reference antibody of the invention for binding to the target, and thus that the test antibody binds to the same or substantially the same epitope or region on the protein αvβ8 integrin, as the reference antibody of the invention. A test antibody that is identified as competing with a reference antibody of the invention in such a method is also an antibody of the present invention. The fact that the test antibody can bind αvβ8 integrin in the same region as the reference antibody of the invention and can compete with the reference antibody of the invention suggests that the test antibody can act as a ligand at the same binding site as the antibody of the invention , and that the test antibody can therefore mimic the action of the reference antibody and is thus an antibody of the invention. This can be confirmed by comparing αvβ8 integrin activity in the presence of a test antibody with αvβ8 integrin activity in the presence of a reference antibody under otherwise identical conditions using an assay that is described in more detail elsewhere herein.

The reference antibody or antigen binding fragment thereof of the invention may be an antibody described herein, for example, an antibody from Table 1 and any variant or fragment thereof as described herein that retains the ability to bind αvβ8 integrin.

As previously stated elsewhere herein, specific binding can be assessed with reference to the binding of an antibody to a molecule that is not a target. This comparison can be made by comparing the ability of an antibody to bind to a target and to another molecule. This comparison can be made as described above in the evaluation of K _D or K _i . The other molecule used in such a comparison can be any molecule that is not the target molecule. Preferably, the other molecule is not identical to the target molecule. Preferably, the target molecule is not a fragment of the target molecule.

The other molecule used to determine specific binding may not be related in structure or function to the target. For example, the other molecule may be an unrelated material or a co-occurring material in the environment.

The other molecule used to determine specific binding may be another molecule involved in the same in vivo pathway as the target molecule, for example, αvβ8 integrin (eg, human αvβ8 integrin). By ensuring that the antibody of the invention has specificity for αvβ8 integrin over another such molecule, unwanted cross-reactivity in vivo can be avoided.

An antibody of the invention may retain the ability to bind to certain molecules that are related to the target molecule.

Alternatively, an antibody of the invention may have specificity for a particular target molecule. For example, it may bind to one target molecule as described herein, but may not bind or may bind with significantly reduced affinity to another target molecule as described herein. For example, full-length mature human αvβ8 integrin may be used as a target, but the antibody that binds to that target may be unable to bind or may bind with less affinity to, for example, other αvβ8 integrin proteins from other species, such as αvβ8 integrin from other species mammals. In some embodiments, the antibody binds to both human and mouse αvβ8 integrin.

An "Fc fusion" protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide. An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner.

A "native sequence Fc region" contains an amino acid sequence identical to the Fc region of an amino acid sequence found in nature. A "variant Fc region" contains an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution relative to the Fc region of the native sequence or the Fc region of the parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in Fc region of the native sequence or in the Fc region of the original polypeptide. The variant Fc region herein preferably has at least about 80% sequence identity with the Fc region of the native sequence and/or the Fc region of the parent polypeptide, and most preferably at least about 90% sequence identity, more preferably at least about 95% at least about 96% at least about 97% at least about 98% at least about 99% sequence identity with them.

As is known in the art, a “constant region” of an antibody refers to the light chain constant region of an antibody or the heavy chain constant region of an antibody, either alone or in combination.

The terms "IgG Fc region", "Fc region", "Fc domain" and "Fc", used interchangeably herein, refer to the portion of the IgG molecule that correlates with the crystallizable fragment produced by the papain digest of the IgG molecule. As used herein, the terms refer to the constant region of the antibody, excluding the first constant region of the immunoglobulin domain, and further refers to portions of this region. Thus, Fc refers to the last two constant regions of the immunoglobulin domains IgA, IgD and IgG and the last three constant regions of the immunoglobulin domains IgE and IgM, and the flexible hinge regions from the N-terminus to these domains or parts thereof. For IgA and IgM, the Fc may include the J chain. For IgG, the Fc contains the immunoglobulin domains Cγ2 and Cγ3 (C gamma 2 and C gamma 3) and the hinge region between Cγ1 (C gamma 1) and Cγ2 (C gamma 2). Although the boundaries of the Fc region may vary, the Fc region of the human IgG heavy chain is generally defined as containing residues C226 or P230 to its carboxyl terminus, numbered according to the Eu index from Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 as described in Kabat et al., 1991. Typically, the Fc domain contains from about amino acid residue 236 to about 447 of the human IgG1 constant domain. An exemplary amino acid sequence of the wild-type human IgG1 Fc domain is shown in SEQ ID NO: 81 and SEQ ID NO: 82 (including the optional terminal lysine (K) residue). The Fc polypeptide may refer to this region when isolated, or to this region in the context of an antibody or an antigen binding fragment or Fc fusion protein thereof.

The heavy chain constant domain contains the Fc region and further contains the CH1 domain and hinge region, as well as the CH2 and CH3 (and optionally CH4 of IgA and IgE) IgG heavy chain domains.

A "functional Fc region" has at least one effector function of a native sequence Fc region. Examples of "effector functions" include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; inhibition of surface cell receptors (eg, B cell receptor), etc. Such effector functions typically require the Fc region to be combined with a binding domain (eg, antibody variable domain or antigen binding fragment thereof) and can be assessed using various known assays known in the art for assessing such antibody effector functions.

A "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The human native sequence Fc regions include the human IgG1 native sequence Fc region (non-A and A allotypes); Fc region of the native sequence of human IgG2; Fc region of the native sequence of human IgG3; and the Fc region of the native human IgG4 sequence, as well as naturally occurring variants thereof.

A "variant Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclasses of receptors, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activating motif (IT AM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are discussed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those that will be identified in the future, are encompassed by the term “FcR” herein.

The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24 :249 (1994)) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997) );Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

"Effect functions" refer to the biological activities related to the Fc region of an antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; inhibition of surface cell receptors (eg, B-cell receptor); and activation of B cells.

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector function(s). Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from a natural source, such as blood.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (eg, NK cells, neutrophils and macrophages), allowing these cytotoxic effectors to cells to specifically bind to antigen-bearing target cells and then destroy the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 from Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay such as those described in US Pat. Nos. 5,500,362 or 5,821,337 or US Pat. No. 6,737,056 (Presta) can be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo , for example, in an animal model such as disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in US Patent No. 7,923,538 and US Patent No. 7,994,290.

An antibody having “enhanced ADCC activity” refers to an antibody that mediates ADCC more effectively in vitro or in vivo compared to the parent antibody, where the antibody and the parent antibody differ in at least one structural aspect, and where the amounts of such antibody and the parent antibody , used in the analysis are essentially the same. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. In some embodiments, ADCC activity is determined using an in vitro ADCC assay as disclosed herein, but other assays or methods for determining ADCC activity are provided, such as in an animal model, etc. In some embodiments, an antibody with increased ADCC activity has increased affinity for Fc gamma RIIIA.

An antibody with “altered” FcR binding affinity or ADCC activity is an antibody that has either increased or decreased FcR binding activity and/or ADCC activity compared to the parent antibody, where the antibody and the parent antibody differ in at least one structural aspect. An antibody that "shows enhanced binding" to an FcR binds at least one FcR with better affinity than the parent antibody. An antibody that "exhibits reduced binding" to an FcR binds at least one FcR with lower affinity than the parent antibody. Such antibodies that exhibit reduced FcR binding may have little or no significant FcR binding, eg, 0-20% FcR binding compared to the Fc region of the native IgG sequence.

“Improved affinity for Fc gamma RIIIA” refers to an antibody that has greater affinity for Fc gamma RIIIA (also called, in some cases, CD 16a) than the parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect.

"Glycoform" refers to a complex oligosaccharide structure containing linkages of various carbohydrate units. Such structures are described in, for example, Essentials of Glycobiology Varki et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), which also provides an overview of standard glycobiology nomenclature. Such glycoforms include, but are not limited to, G2, G1, G0, G-1 and G-2 (see, for example, International Patent Publication No. WO 99/22764).

A “glycosylation profile” is defined as the profile of carbohydrate units that are covalently attached to a protein (eg, a glycoform), as well as the site(s) at which the glycoform(s) are covalently attached to the peptide backbone of a protein, more specifically an immunoglobulin protein.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Antibodies with altered Fc region amino acid sequences and increased or decreased Clq binding ability are described, for example, in US Patent No. 6, 194,551 B l, US Patent No. 7,923,538, US Patent No. 7,994,290 and WO 1999/51642. See also, for example, Idusogie et al., J.

As used herein, the terms “wild-type amino acid,” “wild-type IgG,” “wild-type antibody,” or “wild-type mAb” refer to an amino or nucleic acid sequence that exists naturally in a defined population (e.g., human , mice, rats, in a cage, etc.).

The term “αvβ8 integrin,” as used herein, generally refers to a protein complex containing an alpha integrin subunit (e.g., an alpha-V integrin subunit, e.g., ITGAV, e.g., containing the amino acid sequence SEQ ID NO: 77) and a beta integrin subunit (eg, beta 8 integrin subunit, eg ITGB8, eg containing the amino acid sequence SEQ ID NO: 78). “Human αvβ8 integrin” as used herein generally refers to αvβ8 integrin, e.g., containing the human ITGAV alpha subunit, e.g., containing the sequence SEQ ID NO: 77 and the human ITGB8 beta subunit, e.g., containing the sequence SEQ ID NO: 78. “Mouse αvβ8 integrin” or “mouse αvβ8 integrin” as used herein generally refers to αvβ8 integrin, e.g., containing the mouse ITGAV alpha subunit, e.g., containing the sequence SEQ ID NO: 79, and the mouse ITGB8 beta subunit, e.g., containing sequence SEQ ID NO: 80. The term αvβ8 integrin generally includes homologs and orthologues of αvβ8 integrin, including, but not limited to, human, cynomolgus, rat, rabbit and mouse. As used herein, "αvβ8 integrin" generally refers to a mammalian αvβ8 integrin, e.g., human, rat, mouse, non-human primate, bovine, sheep and porcine αvβ8 integrin (e.g., containing an alpha V integrin subunit and a beta 8 integrin subunit from human, rat, mouse, non-human primate, cow, sheep and pig, respectively). Non-limiting exemplary examples of alphaV integrin subunits include human αvβ8 integrin (see, e.g., Genbank Accession Number P06756.2, SEQ ID NO: 77), cynomolgus monkey (see, e.g., SEQ ID NO:84), and mouse (see ., for example, SEQ ID NO: 79). Non-limiting exemplary examples of beta 8 integrin subunits include human αvβ8 integrin (see, e.g., Genbank Accession Number P26012.1, SEQ ID NO: 78), cynomolgus monkey (see, e.g., SEQ ID NO: 85), and mouse (see eg SEQ ID NO:80). The term "αvβ8 integrin" also includes fragments, variants, isoforms and other homologs of such αvβ8 integrin subunit molecules. Variant αvβ8 integrin molecules are typically characterized by the same type of activity as native αvβ8 integrin, such as the ability to bind an αvβ8 integrin ligand, e.g., as described herein, the ability to induce receptor-mediated activity, and the ability to bind or not bind an antibody or its antigenic fragment according to the invention.

Exemplary amino acid and nucleotide sequences for TGFβ and LAP are known in the art. For example, a polypeptide precursor containing TGFβ1 and LAP (e.g., human sequence, UniProt accession no. P01137) is processed into approximately 30-278 amino acids after translation, UniProt accession no. P01137,corresponding to LAP, and approximately amino acids 279-390 UniProt accession no. P01137, corresponding to human TGFβ1. Also, a precursor polypeptide containing TGFβ3 and LAP (e.g., human sequence, UniProt Accession No. P10600) is processed into approximately 24-300 amino acids after translation, UniProt Accession No. P10600. corresponding LAP and approximately amino acids 301-412 UniProt accession no. 10600, corresponding to human TGFβ3.

αvβ8 integrin may contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, or fifteen or αvβ8 integrin residues that are more accessible on the surface. The target molecule may contain a known epitope from the αvβ8 integrin.

As indicated elsewhere herein, certain positions of the antibody molecule may be altered. By “position” as used herein is meant a location in the sequence of a protein. The positions can be numbered sequentially or according to a specified format, for example, the Eu index and the Cabot index can be used to determine the number of amino acid residues of an antibody. For example, position 297 is a position in a human IgG1 antibody. Relevant positions are determined as described above, usually by matching with other source sequences.

By "residue" as used herein is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also called Asn297, also called N297) is a residue in human IgG1 antibodies.

As is known in the art, “polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to chains of nucleotides of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modification of the nucleotide structure may be made before or after chain assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling moiety. Other types of modifications include, for example, caps, substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, modifications with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), and with charged bonds (e.g. phosphorothioates, phosphorodithioates, etc.), those containing side groups such as, for example, proteins (e.g. nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc. .), those containing intercalators (eg acridine, psoralen, etc.), those containing chelators (eg metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylators , those containing modified linkages (eg, alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups typically present on sugars may be substituted, for example, with phosphonate groups, phosphate groups protected by standard protecting groups, or activated to form additional bonds with additional nucleotides, or conjugated to solid supports. The 5' and 3' terminal OH group may be phosphorylated or replaced by amines or organic capping moieties containing from 1 to 20 carbon atoms. Other hydroxyls can also be converted to standard protecting groups. The polynucleotides may also contain similar forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azidoribose, carbocyclic analogs sugars, alpha or beta anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogues and abasic nucleoside analogues such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR ₂ (“amidate”) ), P(O)R, P(O)OR', CO or CH ₂ ("formacetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing a simple ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide need to be identical. The previous description applies to all polynucleotides described herein, including RNA and DNA.

As used herein, “vector” means a construct that is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors coupled to cationic condensing agents, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells, such as producer cells.

“Host cell” includes a single cell or cell culture that may be or will be the recipient for the vector(s) to incorporate polynucleotide inserts. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells transfected and/or transformed in vivo with a polynucleotide of the present invention.

Host cells can be prokaryotic cells or eukaryotic cells. Examples of eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells such as yeast; plant cells; and insect cells.

Any host cell that is sensitive to cell culture and to the expression of protein or polypeptides can be used in accordance with the present invention. In certain embodiments, the host cell is a mammalian cell. Mammalian cell lines available as expression hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of mammalian cells include, but are not limited to, NS0 cells, HEK 293 cells and Chinese hamster ovary (CHO) cells and their derivatives such as 293-6E and CHO DG44 cells, CHO DXB11 cells and Potelligent® CHOK1SV (BioWa/Lonza , Allendale, NJ). Mammalian host cells also include, but are not limited to, human cervical carcinoma cells (HeLa, ATCC CCL 2), baby hamster kidney cells (BHK, ATCC CCL 10), monkey kidney cells (COS), and human hepatocellular carcinoma cells (eg Hep G2). Other non-limiting examples of mammalian cells that can be used in accordance with the present invention include human retinoblasts (PER.C6®; CruCell, Leiden, The Netherlands); monkey kidney line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293 (HEK 293) or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. 36:59); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); canine kidney cells (MDCK, ATCC CCL 34); gray rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; human hepatoma line (Hep G2); and multiple myeloma cell lines, including, but not limited to, the BALB/c mouse myeloma line (NS0/1, ECACC No: 85110503), NS0 cells and Sp2/0 cells.

Additionally, any number of commercially and non-commercially available cell lines that express polypeptides or proteins can be used in accordance with the present invention. One of ordinary skill in the art will understand that different cell lines may have different nutritional requirements and/or may require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify the conditions as necessary.

The invention includes any eukaryotic expression system known in the art or described herein for producing proteins of interest, such as expression in an insect cell system, a yeast expression system, or a mammalian cell system such as, but not limited to, CHO cells .

As used herein, “expression control sequence” means a nucleic acid sequence that directs transcription of the nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the transcribed nucleic acid sequence.

The terms "leader peptide" or "leader sequence" or "leader signal sequence" or "signal sequence", used interchangeably herein, mean any nucleic acid sequence or amino acid sequence so encoded that may be present at the 5' end of a nucleic acid molecule and/or at or near the N-terminus of the polypeptide, which, if present, may mediate transport of the polypeptide to the destination organelle, including, but not limited to, secretion of the polypeptide from the cell. Such leader sequences include, but are not limited to, nucleic acid sequences containing, for example, ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTCC (SEQ ID NO: 187) and amino acid sequences so encoded, such as, but not limited to, MGWSCIILFLVATATGVHS (SEQ ID NO: 188) . The invention covers these and any other leader signals (nucleic acid and amino acid sequences) known in the art or to be identified that can lead to transport of the polypeptide to the desired organelle, for example, the endoplasmic reticulum and/or secretion from the cell. Typically, the signal peptide is removed and/or absent from the mature polypeptide.

As used herein, “treatment” is an approach to achieving positive or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: improved survival (decreased mortality), reduced inflammatory response to disease, reduced degree of tissue fibrosis, improved appearance of disease lesions, limitation of pathological lesions lesions at focal sites, reducing the extent of damage from the disease, reducing the duration of the disease and/or reducing the number, extent or duration of symptoms associated with the disease. The term includes the administration of compounds or agents of the present invention to prevent or delay the onset of symptoms, complications or biochemical signs of a disease, alleviate symptoms or stop or inhibit the progression of a disease, condition or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease or to prevent the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms after the disease has manifested. In some embodiments, the disease, condition, or disorder is cancer.

As used herein, the term “cancer” includes all types of cancers or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. Examples of cancer disorders include, but are not limited to, solid tumors, hematologic cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, such as sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas), of various organ systems such as the liver, lung, breast, lymph, gastrointestinal tract (eg, colon), genitourinary tract (eg, kidneys, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung carcinoma, small intestinal cancer, and esophageal cancer. Squamous cell carcinomas include malignant neoplasms, for example , in the lungs, esophagus, skin, head and neck area, mouth, anus and cervix. In one embodiment, the cancer is melanoma, such as advanced melanoma. Metastatic lesions of the above-mentioned cancers can also be treated using the methods and compositions of the present invention. Exemplary cancers that can be treated, eg, reduced in growth, using the antibody molecules described herein include cancers that are typically sensitive to immunotherapy.

“Relief” means a reduction or improvement in one or more symptoms compared to not taking the anti-αvβ8 integrin antibody. “Relief” also includes shortening or reducing the duration of a symptom.

As used herein, an “effective dosage” or “effective amount” of a drug, compound or pharmaceutical composition is an amount sufficient to effect one or more beneficial or desired results. In more specific aspects, the effective amount prevents, reduces or alleviates the symptoms of a disease, such as cancer, and/or prolongs the survival of the subject being treated. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reduction of one or more symptoms of a disease, disorder or condition mediated by αvβ8 integrin, reduction of the dose of other drugs needed to treat the disease, enhancement of the effect of another drug, and/or slowing of progression diseases in patients. An effective dose can be administered in one or more administrations. For purposes of this invention, an effective dosage of a drug, compound or pharmaceutical composition is an amount sufficient to provide prophylactic or therapeutic treatment directly or indirectly. As understood in a clinical context, an effective dosage of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of the administration of one or more therapeutic agents, and one agent may be considered to be administered in an effective amount if, in combination with one or more other agents, the desired result can be or is achieved.

Antibodies or an antigen binding fragment thereof can be administered in combination with one or more therapies (eg, referred to herein as a “second therapy”). By “in combination with” it is not meant that the therapy or therapeutic agents must be administered simultaneously and/or formulated for co-delivery, although these modes of delivery are within the scope described herein. Anti-αvβ8 integrin antibodies or an antigen-binding fragment thereof can be administered simultaneously, before or after one or more other additional therapies or therapeutic agents. The anti-αvβ8 integrin antibody or antigen binding fragment thereof and the second therapy, such as another agent or therapeutic protocol, can be administered in any order. In general, each agent will be administered at a dose and/or schedule specific to that agent. It should also be understood that the additional therapeutic agent used in this combination may be administered together in a single composition or administered separately in different compositions. In some embodiments, the doses used in combination will be lower than the doses used individually.

A “synergistic combination” or a combination that acts “synergistically” is a combination that exhibits enhanced effects that are not predicted when compared to the merely additive effect of the individual combination therapies.

The "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals (eg, cows, pigs, horses, chickens, etc.), sporting animals, pets, primates, horses, dogs, cats, mice and rats. In some embodiments, the individual is at risk for a disease, disorder, or condition mediated by or associated with αvβ8 integrin binding and signaling mediated by it. In certain embodiments, the subject has a disorder or condition described herein, such as cancer.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil/water emulsion, and various types of wetting agents. The preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBSA) or normal (0.9%) saline. Compositions containing such carriers are formulated by known, commonly used methods (see, for example, Remington's Pharmaceutical Sciences, ^18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy ^20th Ed. Mack Publishing, 2000).

Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting.

II. ANTI- α v β 8 INTEGRIN ANTIBODIES

The present invention relates to antibodies and antigen-binding fragments thereof that bind to αvβ8 integrin. Preferably, the antibodies specifically bind to αvβ8 integrin, i.e., they bind to αvβ8 integrin, but they do not bind detectably or bind with lower affinity to other αv integrins (eg, αvβ3 integrin, αvβ5 integrin and αvβ6 integrin). The invention further relates to anti-αvβ8 integrin antibodies that exhibit altered effector function. In some embodiments, the altered effector function is reduced ADCC. In some embodiments, the altered effector function is reduced CDC. The invention also relates to compositions containing such antibodies, as well as to the use of such antibodies, including therapeutic and pharmaceutical uses.

In one embodiment, the description provides any of the following or compositions (including pharmaceutical compositions) containing an antibody having a light chain or fragment thereof and a heavy chain or fragment thereof derived from, but not identical to, the mouse hybridoma antibody ADWA-11 (also referred to as ADWA11, mADWA11, mADWA-11), as described in US Patent No. 9,969,804, which is incorporated herein by reference in its entirety, and as shown in, for example , SEQ ID NOs: 20-33 and 71-76 of the present specification.

Antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab', F(ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins containing an antibody fragment (e.g., antibody domain), humanized antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site with the required specificity, including glycosylated antibody variants, amino acid sequence variants antibodies and covalently modified antibodies. Antibodies may be derived from mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the anti-αvβ8 integrin antibody is a monoclonal antibody. In some embodiments, the anti-αvβ8 integrin antibody is a human antibody or a humanized antibody. In some embodiments, the anti-αvβ8 integrin antibody is a chimeric antibody.

The anti-αvβ8 integrin antibodies of the invention can be prepared by any method known in the art. General techniques for producing human and mouse antibodies are known in the art and/or described herein.

Following initial identification, the activity of a candidate anti-αvβ8 integrin antibody can be further confirmed and refined by bioassays known to test the target biological activity. In some embodiments, an in vitro cell-based assay is used to further characterize a candidate anti-αvβ8 integrin antibody. For example, bioassays can be used to directly screen candidates. Several methods for identifying and characterizing anti-αvβ8 integrin antibodies are described in detail in the Examples.

Table 1 below provides summarized amino acid and nucleotide sequence data for murine, chimeric and humanized anti-αvβ8 integrin antibodies, for example, as described herein. The amino acid and nucleotide sequences of the heavy and light chain CDRs, the amino acid and nucleotide sequences of the heavy and light chain variable region, and the amino acid and nucleotide sequences of the heavy and light chains are shown in this table. In general, unless specifically stated, the anti-αvβ8 integrin antibodies of the invention may include any combination of one or more Kabat CDRs and/or Chotia hypervariable loops as shown in Table 1. In some embodiments, the anti-αvβ8 integrin antibodies of the invention may include any combination of one or more VH and/or VL sequences as shown in Table 1. In some embodiments, anti-αvβ8 integrin antibodies of the invention may include any combination of one or more framework regions (e.g., FR1, FR2, FR3 and FR4) as described in Table 1. As is generally understood and as indicated in Table 1, each VH and VL sequence typically includes three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4.

In some embodiments, where the anti-αvβ8 integrin antibody contains a C-terminal lysine (K) amino acid residue of the heavy chain of the polypeptide (e.g., the human IgG1 heavy chain contains a terminal lysine), one of ordinary skill in the art will appreciate that the lysine residue may be truncated, yielding an antibody with a heavy chain lacking a C-terminal lysine residue. Additionally, the antibody heavy chain can be produced using a nucleic acid that does not encode lysine. Thus, in some embodiments, the anti-αvβ8 integrin antibody contains a heavy chain where the terminal lysine, present in other cases, is not present.

Table 1: Amino acid and nucleotide sequences for α v β 8 integrin antibodies and other peptides.

Name SEQ ID NO. Subsequence ADWA11 2.4 VL
VL amino acid sequence
The underlined amino acid residues are CDR sequences according to Kabat (also called ADWA11_VK01_2.4) 7 diqmtqspsslsasvgdrvtitcrstkslshfngntylfwyqqkpgkapkrliyymsslasgvpsrfsgsgsgtdftltisslqpedfatyycqqsleypftfgggtkveik ADWA11 2.4 CDR-L1 according to Kabat eleven rstkslshfngntylf ADWA11 2.4 CDR-L2 according to Kabat 12 yYmsslas ADWA11 2.4 CDR-L3 according to Kabat 13 qqsleypft ADWA11 2.4 CDR-L1 by Chotia 17 stkslshfngntyl ADWA11 2.4 CDR-L2 by Chotia 18 yymss ADWA11 2.4 CDR-L3 by Chotia 19 qsleypft ADWA11 2.4 VH
VH amino acid sequence
The underlined amino acid residues are CDR sequences according to Kabat 6 evqlvesggglvqpggslrlscaasgfnikdyymnwvrqapgkglewvgwidpdqgntiyepkfqgrftisadtsknsaylqmnslraedtavyycarrllmdywgqgtlvtvss ADWA11 2.4 CDR-H1 according to Kabat 8 dyymn ADWA11 2.4 CDR-H2 according to Kabat 9 widpdqgntiyepkfqg ADWA11 2.4 CDR-H3 according to Kabat 10 rllmdy ADWA11 2.4 CDR-H1 by Chotia 14 gfnikdyymn ADWA11 2.4 CDR-H2 by Chotia 15 widpdqgn ADWA11 2.4 CDR-H3 by Chotia 16 rllmdy ADWA11 2.4
Light chain (LC) amino acid sequence
VL sequence is underlined 5 diqmtqspsslsasvgdrvtitcrstkslshfngntylfwyqqkpgkapkrliyymsslasgvpsrfsgsgsgtdftltisslqpedfatyycqqsleypftfgggtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskds tyslsstltlskadyekhkvyacevthqglsspvtksfnrgec ADWA11 2.4
Heavy chain amino acid sequence (HC)
VH sequence is underlined 2 evqlvesggglvqpggslrlscaasgfnikdyymnwvrqapgkglewvgwidpdqgntiyepkfqgrftisadtsknsaylqmnslraedtavyycarrllmdywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysls svvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtkn qvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk ADWA11 2.4
Heavy chain amino acid sequence without terminal lysine residue
VH sequence is underlined 3 evqlvesggglvqpggslrlscaasgfnikdyymnwvrqapgkglewvgwidpdqgntiyepkfqgrftisadtsknsaylqmnslraedtavyycarrllmdywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysls svvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtkn qvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg ADWA11 2.4
Light chain DNA sequence
Nucleic acid residues encoding VL are underlined
Leader-encoding nucleic acid residues are indicated in capital letters 4 atgggatggagctgtatcatcctcttcttggtagcaacagctacaggcgtgcactccGACATCCAGATGACCCAGTCCCCTTCCAGCCTGAGCGCTTCCGTGGGCGACAGGGTGACCATCACCTGCAGGTCCACCAAGTCCCTGTCCCACTTCAACGGCAACACCTACCTGTTCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGAGGCTGATCTACTACATGTCCTCCCTGGCCTC CGGAGTGCCCTCCAGGTTCTCCGGATCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCCCTGGAGTACCCCCTTCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTC TATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT ADWA11 2.4
Light chain DNA sequence
Nucleic acid residues encoding VL are underlined 185 GACATCCAGATGACCCAGTCCCCTTCCAGCCTGAGCGCTTCCGTGGGCGACAGGGTGACCATCACCTGCAGGTCCACCAAGTCCCTGTCCCACTTCAACGGCAACACCTACCTGTTCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGAGGCTGATCTACTACATGTCCTCCCTGGCCTCCGGAGTGCCCTCCAGGTTCTCCGGATCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTC CCTGCAGCCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCCCTGGAGTACCCCCTTCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT ADWA11 2.4
DNA sequence VL 186 GACATCCAGATGACCCAGTCCCCTTCCAGCCTGAGCGCTTCCGTGGGCGACAGGGTGACCATCACCTGCAGGTCCACCAAGTCCCTGTCCCACTTCAACGGCAACACCTACCTGTTCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGAGGCTGATCTACTACATGTCCTCCCTGGCCTCCGGAGTGCCCTCCAGGTTCTCCGGATCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTC CCTGCAGCCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCCCTGGAGTACCCCTTTCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAA ADWA11 2.4
Light chain and heavy chain DNA leader sequences 187 ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTCC ADWA11 2.4
Leader amino acid sequences of the light chain and heavy chain 188 MGWSCIILFLVATATGVHS ADWA11 2.4
Heavy chain DNA sequence (lysine-terminal)
Nucleic acid residues encoding VH are underlined
Leader-encoding nucleic acid residues are indicated in capital letters 1 atgggatggagctgtatcatcctcttcttggtagcaacagctacaggcgtgcactccGAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAACACCATCTACGA GCCCAAGTTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTTC AAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGCTGGGGCACCGTCAGTCTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG TGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGAAAA ADWA11 2.4
Heavy chain DNA sequence (lysine-terminal)
Nucleic acid residues encoding VH are underlined 189 GAGGTGCAGCTGGTGGAAAGCGGAGGAGGGCCTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAACACCATCTACGAGCCCAAGTTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGC AGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGA TCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGAAAA ADWA11 2.4
DNA sequence VH 190 GAGGTGCAGCTGGTGGAAAGCGGAGGAGGGCCTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAACACCATCTACGAGCCCAAGTTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGC AGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCA ADWA11 2.4
DNA heavy chain sequence
Nucleic acid without terminal lysine, VH-coding residues are underlined
Leader-encoding nucleic acid residues are indicated in capital letters 183 atgggatggagctgtatcatcctcttcttggtagcaacagctacaggcgtgcactccGAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAACACCATCTACGA GCCCAAGTTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTTC AAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGCTGGGGCACCGTCAGTCTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG TGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA ADWA11 2.4
DNA heavy chain sequence
Nucleic acid without terminal lysine, VH-coding residues are underlined 191 GAGGTGCAGCTGGTGGAAAGCGGAGGAGGGCCTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAACACCATCTACGAGCCCAAGTTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGC AGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGA TCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA Mouse hybridoma antibody ADWA-11
VL amino acid sequence
The underlined amino acid residues are CDR sequences according to Kabat 21 DIVMTQAAPSVPVTPGESVSISCRSTKSLLHFNTYLFWFLQRPGQSPQRLIYYMSNLASGVPDRFSGRGSGTDFTLRISRVEAEDVGVYYCMQSLEYPFTFGTGTKLEIK Mouse hybridoma antibody ADWA-11
VH amino acid sequence
The underlined amino acid residues are CDR sequences according to Kabat 20 EVQLQQSGAELVRPGAFVKLSCKASGFNIKDYYMNWVLQRPEQGLEWIGWIDPDNGNTIYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCARRLLMDYWGQGTSVTVSS Mouse hybridoma antibody ADWA-11
CDR-L1 according to Kabat 25 RSTKSLLHFNGNTYLF Mouse hybridoma antibody ADWA-11
CDR-L2 according to Kabat 26 YYMSNLAS Mouse hybridoma antibody ADWA-11
CDR-L3 according to Kabat 27 MQSLEYPFT Mouse hybridoma antibody ADWA-11 Alternative
Alternative CDR-L1 according to Kabat 71 rstksllhfngntylf Mouse hybridoma antibody ADWA-11 Alternative
Alternative CDR-L2 according to Kabat 72 yymsnlas Mouse hybridoma antibody ADWA-11 Alternative
Alternative CDR-L3 according to Kabat 73 mqsleypft Mouse hybridoma antibody ADWA-11
CDR-H1 according to Kabat 22 DYYMN Mouse hybridoma antibody ADWA-11
CDR-H2 according to Kabat 23 WIDPDNGNTIYDPKFQG Mouse hybridoma antibody ADWA-11
CDR-H3 according to Kabat 24 RLLMDY Mouse hybridoma antibody ADWA-11
CDR-L1 by Chotia 31 STKSLLHFNGNTYL Mouse hybridoma antibody ADWA-11
CDR-L2 by Chotia 32 YYMSN Mouse hybridoma antibody ADWA-11
CDR-L3 by Chotia 33 QSLEYPFT Mouse hybridoma antibody ADWA-11 Alternative
Alternative CDR-L1 according to Chotia 74 stksllhfngntyl Mouse hybridoma antibody ADWA-11 Alternative
Alternative CDR-L2 according to Chotia 75 yymsn Mouse hybridoma antibody ADWA-11 Alternative
Alternative CDR-L3 according to Chotia 76 qsleypft Mouse hybridoma antibody ADWA-11
CDR-H1 by Chotia 28 GFNIKDYYMN Mouse hybridoma antibody ADWA-11
CDR-H2 by Chotia 29 WIDPDNGN Mouse hybridoma antibody ADWA-11
CDR-H3 by Chotia thirty RLLMDY ADWA11_VK01 (1) (also referred to herein as adwa_VL_1.1 L46R)
VL amino acid sequence 47 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_1a (1) L29I
VL amino acid sequence 48 DIQMTQSPSSLSSASVGDRVTITCRSTKSILHFNGNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_1b (1) L30S
VL amino acid sequence 49 DIQMTQSPSSLSSASVGDRVTITCRSTKSLSHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_1c (1) T36S
VL amino acid sequence 50 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNGNSYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_2a (1) Y55A
VL amino acid sequence 51 DIQMTQSPSSSLSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYAMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_2b (1) M56A
VL amino acid sequence 52 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_2c (1) N58S
VL amino acid sequence 53 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_2d (1) A60Q
VL amino acid sequence 54 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_3a (1) M94Q
VL amino acid sequence 55 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLEYPFTFGQGTKVEIK ADWA11_VK01_3b (1) L97Y
VL amino acid sequence 56 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSYEYPFTFGQGTKVEIK ADWA11_VK01_3c (1) E98S
VL amino acid sequence 57 DIQMTQSPSSSLSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLSYPFTFGQGTKVEIK ADWA11_VK01_3d (1) Y99T
VL amino acid sequence 58 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLETPFTFGQGTKVEIK ADWA11_VK01_4a (1) F101L
VL amino acid sequence 59 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPLTFGQGTKVEIK ADWA11_VK01_4b (1) F101W
VL amino acid sequence 60 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPWTFGQGTKVEIK ADWA11_VK01_4c (1) Q105G
VL amino acid sequence 61 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGGGTKVEIK ADWA11VK1 IGKV2-28
VL amino acid sequence 62 DIVMTQSPLSLPVTPGEPASISCRSTKSLLHFNTYLFWYLQKPGQSPQLLIYYMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK2 IGKV2-30
VL amino acid sequence 63 DVVMTQSPLSLPVTLGQPASISCRSTKSLLHFNGNTYLFWFQQRPGQSPRRLIYYMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK3 IGKV4-1
VL amino acid sequence 64 DIVMTQSPDSLAVSLGERATINCRSTKSLLHFNGNTYLFWYQQKPGQPPKLLIYYMSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK4 IGKV1-39
VL amino acid sequence 65 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKLLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11VK5 IGKV3-11
VL amino acid sequence 66 EIVLTQSPATLSLSPGERATLSCRSTKSLLHFNGNTYLFWYQQKPGQAPRLLIYYMSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_2.1
VL amino acid sequence 67 diqmtqspsslsasvgdrvtitcrstkslshfngntylfwyqqkpgkapkrliyymsnlasgvpsrfsgsgsgtdftltisslqpedfatyycqqsleypftfgggtkveik ADWA11_VK01_2.2
VL amino acid sequence 68 DiqmtqspsslsasvgdrvtitcrstkslshfngntylfwyqqkpgkapkrliyymsnlasgvpsrfsgsgsgtdftltisslqpedfatyycMqsyeypftfgggtkveik ADWA11_VK01_2.3
VL amino acid sequence 69 diqmtqspsslsasvgdrvtitcrstkslshfngntylfwyqqkpgkapkrliyyAsnlasgvpsrfsgsgsgtdftltisslqpedfatyycqqsleypftfgggtkveik ADWA11VH1 IGHV1-46
VH amino acid sequence 34 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMNWVRQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH2 IGHV3-23
VH amino acid sequence 35 EVQLLESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH3 IGHV3-30
VH amino acid sequence 36 QVQLVESGGGVVQPGRSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH4 IGHV1-69
VH amino acid sequence 37 QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYMNWVRQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 IGHV3-48
VH amino acid sequence 38 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11 VH05_VK1 (also referred to herein as adwa_VH_1.5 T28N+F29I+R72A+A49G+L79A+N74T+A75S)
VH amino acid sequence 39 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 D61E
VH amino acid sequence 40 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYEPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 N55Q
VH amino acid sequence 41 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDQGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 N28Q
VH amino acid sequence 42 EVQLVESGGGLVQPGGSLRLSCAASGFQIKDYYMNWVRQAPGKGLEWVGWIDPDQGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5K30A
VH amino acid sequence 43 EVQLVESGGGLVQPGGSLRLSCAASGFNIADYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 N57Q
VH amino acid sequence 44 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGQTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 P62A
VH amino acid sequence 45 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDAKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 K63A
VH amino acid sequence 46 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPAFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS Typical amino acid sequence of human integrin alpha V (ITGAV) subunit 77 mllgtlllilyilmlcrmfllvgapkanttqpgiveggqvlkcdwsstrrcqpiefdatgnrdyakddplefkshqwfgasvrskqdkilacaplyhwrtemkqerepvgtcflqdgtktveyapcrsqdidadgqgfcqggfsidftkadrvllggpgsfywqgqlisdqvaeivskydpn vysikynnqlatrtaqaifddsylgysvavgdfngdgiddfvsgvpraartlgmvyiydgknmsslynftgeqmaayfgfsvaatdingddyadvfigaplfmdrgsdgklqevgqvsvslqrasgdfqttklngfevfarfgsaiaplgdldqdgfndiaiaapyggedkkgivyifngrstgln avpsqilegqwaarsmppsfgysmkgatdidkngypdlivgafgvdrailyrarpvitvnaglevypsilnqdnktcslpgtalkvscfnvrfclkadgkgvlprklnfqvellldklkqkgairralflysrspshsknmtisrgglmqceeliaylrdesefrdkltpitifmeyrldyrtaadttglqpilnqft pani edvgpvvqhiyelrnngpssfskamlhlqwpykynnntllyilhydidgpmnctsdmeinplrikisslqttekndtvagqgerdhlitkrdlalsegdihtlgcgvaqclkivcqvgrldrgksailyvksllwtetfmnkenqnhsyslkssasfnviefpyknlpieditnstlvttnvtwgiqpapmp vpvwviilavlagllllavlvfvmyrmgffkrvrppqeeqereqlqphengegnset Typical amino acid sequence of human integrin beta 8 (ITGB8) subunit 78 MCGSALAFFTAAFVCLQNDRRGPASFLWAAWVFSLVLGLGQGEDNRCASSNAASCARCLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEINTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIH NQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANNLNNLVVEAYQKLISEVKVQVENQVQGIYFNITAICPD GSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCHFDEDQFSSESCKSHKDQPVCSGRGVCVCGKCSCHKIKLGKVYGKYCEKDDFSCPYHHGNLCAGHGECEAGRCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTC VCGRCECTDPRSIGRFCEHCPTCYTACKENWNCMQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAHETFRCNF Typical amino acid sequence of mouse integrin alpha V (ITGAV) subunit 79 MAAPGRLLLRPRPGGLLLLLPGLLLPLADAFNLDVESPAEYAGPEGSYFGFAVDFFEPSTSSRMFLLVGAPKANTTQPGIVEGGQVLKCECSSSRRCQPIEFDSTGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSKNIDADGQGFCQGGFSIDFTKADRVLLGGPG SFYWQGQLISDQVAEIISKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIEDFVSGVPRAARTLGMVYIYDGKNMSSLHNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRAVGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGED KKGLVYIFNGRSTGLNSVPSQILEGQWAAQSMPPSFGYSMKGATDVDRNGYPDLVVGAFGVDRAVLYRARPVVTVNAGLEVYPSILNQDNKICPLPGTALKVSCFNVRFCLKADGKGTLPRKLHFQVELLLDKLKQKGAIRRALFLHNRSPVHSKTMTVFRGGQMQCEELVAYLRDESEFRDKLTPITIFMEYRLDQR TAADATGLQPILNQFTPANVSRQAHILLDCGEDNVCKPKLEVSVNSDQKKIYIGDDNPLTLTVKAQNQGEGAYEAELIVSIPPQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLKIQSSNSFDNVSPVVSYKVDLAVLAAVEIRGVSSPDHIFLPIPNWEYKENPE TEEDVGPIVQHIYELRNNGPSSFSKAILNLQWPYKYNNNTLLYILHYDIDGPMNCTADTEINPLRIKTPEKNDTAAAGQGERNHLITKRDLTLREGDVHTLGCGIAKCLQITCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNIIEFPYKNLPIEDLFNSTLVTTNITWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVM YRMGFFKRVRPPQEEQEREQLQPHENGEGNSET Typical amino acid sequence of mouse integrin beta 8 (ITGB8) subunit 80 MCGSALAFLTAALLSLHNCQRGPALVLGAAWVFSLVLGLGQSEHNRCGSANVVSCARCLQLGPECGWCVQEDFVSGGSGSERCDTVSSLISKGCPVDSIEYLSVHVVTSSENEINTQVTPGEVSVQLHPGAEANFMLKVRPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSKKMALYSRDFRLGFGSYVDKTVSPYISIH PERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGAIAGEIESKAANNLNNLVVEAYKKIISEVKVQLENQVHGVHFNI TAICPDGARKPGISGCGNVTSNDEVLFNVTVVMKTCDIMGGKNYAIIKPIGFNETTKVHIHRSCSCQCENHRGLKGQCAEAAPDPKCPQCDDSRCHFDEDQFPSETCKPQEDQPVCSGRGVCICGKCLCHKTKLGRVYGQYCEKDDFSCPYLHGDVCAGHGECEGGRCQCFSGWEGDRCQCPSASAQHCVNSKGQVCSGRG TCVCGRCECTDPRSIGRLCEHCPTCHLSCSENWNCLQCLHPHNLSQAALDQCKSSCAVMEQHRMDQTSECLSGPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNNNKIKSSSSDYRMSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAQEAFRCNF Typical amino acid sequence of cynomolgus integrin alpha V (ITGAV) subunit 84 maspprrrlrlgprglplllsglllplcrafnldvdspaeysgpegsyfgfavdffvpsassrmfllvgapkanttqpgiveggqvlkcdwsstrrcqpiefdatgnrdyakddplefkshqwfgasvrskqdkilacaplyhwrtelkqerepvgtcflqdgtktveyapcrsqdidadgqgfcq ggfsidftkadrvllggpgsfywqgqlisdqvaeivskydpnvysikynnqlatrtaqaifddsylgysvavgdfngdgiddfvsgvpraartlgmvyiydgknmssiynftgdqmaayfgfsvaatdingddyadvfigaplfmdrgsdgklqevgqvsvslqrasgdfqttklngfev distant gglmqceeliaylrdesefrdkltpitifmeywldyrtaadttglqpilnqftpanisrqahilldcgednvckpklevfvdsdqkkiyigddnpltlivkaqnqgegayeaelivsiplqadfigvvrnsealarlscafktenqtrqvvcdlgnpmkagtqllaglrfsvhqqsemdtsvkfdlqiqs snlfdkvspvvshkvdlavlaaveirgvsspdhiflpipnwehkenpeteedvgpvvqhiyelrnngpssfskamlhlqwpykynnntllyilhydidgpmnctsdmeinplrikisslqatekndtvagqgerdhlitkrdlalsegdihtlgcgvaqclkivcqvgrldrgksailyvksllwtetfmnkenq nhsyslkssasfnviefpyknlpieditnstlvttnvtwgiqpapmpvpvwviilavlagllllavlvfvmyrmgffkrvrppqeeqereqlqphengegnset Typical amino acid sequence of cynomolgus integrin subunit beta 8 (ITGB8) 85 mcgsalafftaafvclqndrrgpasflwaawvlslvlglgqgednicassnaascarclalgpecgwcvqedfisggsrsercdivsnliskgcsvdsieypsvhviipteneintqvtpgevsiqlrpgaeanfmlkihplkkypvdlyylvdvsasmhnnieklnsvgndlsrkmaffsrdfrlgfgsyv dktvspyisihperihnqcsdynldcmpphgyihvlsltenitefekavhrqkisgnidtpeggfdamlqaavceshigwrkeakrlllvmtdqtshlaldsklagivvpndgnchlknnvyvksttmehpslgqlseklidnninvifavqgkqfhwykdllpllpgtiageieskaanlnnlvveayqklisevkv hvenqvqgvyfnitaicpdgsrkpgmegcrnvtsnhevlfnvtvtmkkcdvtggknyaiikpigfnetakihihrncscqcednrgpkgkcvdetfldskcfqcdenkchfdedqfssesckshkdqpvcsgrgvcvcgkcschkiklgkvygkycekddfscpyhhgnlcaghgeceagrcq cfsgwegdrcqcpsaaaqhcvnskgqvcsgrgtcvcgrcectdprsigrfcehcptchtackenwncvqclhphnlsqaildqcktscalmeqqhyvdqtsecfsspsylriffiifivtfligllkvliirqvilqwnsnkiksssdyrvsaskkdklilqsvctravtyrrekpeeikmdisklnahet frcnf Typical wild-type human IgG1 Fc (includes portion of _CH1 and hinge region, _CH2 and _CH3 )
The effector function of the wild-type LLGG sequence is in italics.
NST Asn297 N-linked glycosylation site 81 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Typical human IgG1 constant region
The effector function of the wild-type LLGG sequence is in italics.
NST Asn297 N-linked glycosylation site
Includes terminal lysine 82 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Typical human IgG1 constant region without effectors 184 astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnakt kpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg Typical human IgG1 constant region nucleic acid sequence without effectors 192 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAC CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA Typical human IgG1 constant region (lysine-terminal) 181 astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaagapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnakt kpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgK Typical human IgG1 constant region nucleic acid sequence (with terminal lysine) 193 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGAC CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGAAAA Typical human IgG2 constant region 70 astkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdkterkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpree qfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk Typical human light chain kappa constant region (Cκ) 83 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Typical human light chain kappa constant region (Cκ) nucleic acid sequence 194 CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Typical mouse IgG1 heavy chain constant region 86 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPPVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKAFAC AVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTTCSVLHEGLHNHHTEKSLSHSPGK Typical mouse light chain constant region 87 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSSPIVKSFNRNEC adwa_VH_1.1 T28N+F29I 88 EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVRQAPGKGLEWVAWIDPDNGNTIYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS adwa_VH_1.2
T28N+F29I+R72A 89 EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVRQAPGKGLEWVAWIDPDNGNTIYDPKFQGRFTISaDNAKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS adwa_VH_1.3
T28N+F29I+R72A+A49G+L79A 90 EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVRQAPGKGLEWVgWIDPDNGNTIYDPKFQGRFTISaDNAKNSaYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS adwa_VH_1.4
T28N+F29I+R72A+N74T+A75S 91 EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVRQAPGKGLEWVAWIDPDNGNTIYDPKFQGRFTISaDtsKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS adwa_VL_1.2
L46R+Y36F 92 DIQMTQSPSSSLSASVGDRVTITCRSTKSLLHFNGNTYLFWfQQKPGKAPKrLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK VH05-2(F64V)VK01 93 evqlvesggglvqpggslrlscaasgfnikdyymnwvrqapgkglewvgwidpdqgntiyepkVqgrftisadtsknsaylqmnslraedtavyycarrllmdywgqgtlvtvss 5662_01 94 EPKFQGRFTISADTS 5662_02 95 TAVYYCARRLLMDYW 5662_03 96 TAVYYSARRLLXDYW 5662_04 97 KSLLHFNGNTYLFWY 5662_05 98 PKRLIYYMSNLASGV 5662_06 99 PKRLIYYXSNLASGV 5662_07 100 LIYYMSNLASGVPSR 5662_08 101 LIYYXSNLASGVPSR 5662_09 102 FATYYCMQSLEYPFT 5662_10 103 FATYYSXQSLEYPFT 5662_11 104 EYPFTFGQGTKVEIK 5662_12 105 EPKVQGRFTISADTS 5662_13 106 KSLSHFNGNTYLFWY 5662_14 107 FATYYCQQSLEYPFT 5662_15 108 FATYYSQQSLEYPFT 5662_16 109 FATYYCMQSYEYPFT 5662_17 110 FATYYSXQSYEYPFT 5662_18 111 EYPFTFGGGTKVEIK 5662_19 112 KRLIYYASNLASGVP 5662_20 113 KRLIYYMSSLASGVP 5662_21 114 KRLIYYXSSLASGVP 5662_22 115 QGDSLRTYYASWYQQ 5662_23 116 VLVIYGKHKRPSGIP 5662_24 117 EADYYCMSRSIWGNP 5662_25 118 EADYYSXSRSIWGNP 5662_26 119 SETLSLTCAVSGYST 5662_27 120 GLEWIGSISHTGNTY 5662_28 121 NPPLKSRVTISVDTS 5662_29 122 DTAVVYCARGGGISR ADWA 11 VK01 123 DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC ADWA11 VH05 124 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ADWA11 VH05 without terminal lysine residue 182 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Wild-type human IgG1 hinge region 125 EPKSCDKTHTCPPCPAPELLGGP Effector null (3m, triple mutated) variant of the human IgG1 hinge region 126 EPKSCDKTHTCPPCPAPEAAGAP IGHV3-07 (DP-54) germline heavy chain 127 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR IGKV1-39 (DPK-9) germline light chain 128 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP Synthetic HA peptide derived from influenza A hemagglutinin 129 PKYVKQNTLKLAT TET 830 modified/T helper epitope from tetanus toxin 130 AQYIKANSKFIGITEL IMGT - heavy chain 195 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMSWVRQA PGKGLEWVAN IKQDGSEKYY
VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR IMGT - light chain 196 DIQMTQSPSS LSASVGDRVT ITCRASQSIS ----S-YLNW YQQKPGKAPK LLIYAASSLQ
SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCQQSYSTP

*In some peptides, methionine is replaced by norleucine.

Table 14: Typical heavy chain CDRs according to Kabat

VH (SEQ ID NO) CDR-H1 (SEQ ID NO) CDR-H2 (SEQ ID NO) CDR-H3 (SEQ ID NO) ADWA-11 VH mouse (SEQ ID NO: 20) DYYMN (SEQ ID NO: 22) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) RLLMDY (SEQ ID NO: 24) ADWA11 2.4 VH (SEQ ID NO: 6) dyymn (SEQ ID NO: 8) widpdqgntiyepkfqg (SEQ ID NO: 9) rllmdy (SEQ ID NO: 10) ADWA11VH1 IGHV1-46(SEQ ID NO: 34) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDQKFQG (SEQ ID NO: 157) rllmdy (SEQ ID NO: 10) ADWA11VH2 IGHV3-23(SEQ ID NO: 35) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDDSVKG (SEQ ID NO: 158) rllmdy (SEQ ID NO: 10) ADWA11VH3 IGHV3-30 (SEQ ID NO: 36) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDDSVKG (SEQ ID NO: 158) rllmdy (SEQ ID NO: 10) ADWA11VH4 IGHV1-69 (SEQ ID NO: 37) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDQKFQG (SEQ ID NO: 157) rllmdy (SEQ ID NO: 10) ADWA11VH5 IGHV3-48 (SEQ ID NO: 38) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDDSVKG (SEQ ID NO: 158) rllmdy (SEQ ID NO: 10) ADWA11 VH05_VK1 (also referred to herein as adwa_VH_1.5 T28N+F29I+R72A+A49G+L79A+N74T+A75S) (SEQ ID NO: 39) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) rllmdy (SEQ ID NO: 10) ADWA11VH5 D61E (SEQ ID NO: 40) dyymn (SEQ ID NO: 8) WIDPDNGNTIYEPKFQG (SEQ ID NO: 160) rllmdy (SEQ ID NO: 10) ADWA11VH5 N55Q (SEQ ID NO: 41) dyymn (SEQ ID NO: 8) WIDPDQGNTIYDPKFQG (SEQ ID NO: 161) rllmdy (SEQ ID NO: 10) ADWA11VH5 N28Q (SEQ ID NO: 42) dyymn (SEQ ID NO: 8) WIDPDQGNTIYDPKFQG (SEQ ID NO: 161) rllmdy (SEQ ID NO: 10) ADWA11VH5K30A (SEQ ID NO: 43) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) rllmdy (SEQ ID NO: 10) ADWA11VH5 N57Q (SEQ ID NO: 44) dyymn (SEQ ID NO: 8) WIDPDNGQTIYDPKFQG (SEQ ID NO: 162) rllmdy (SEQ ID NO: 10) ADWA11VH5 P62A (SEQ ID NO: 45) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDAKFQG (SEQ ID NO: 163) rllmdy (SEQ ID NO: 10) ADWA11VH5 K63A (SEQ ID NO: 46) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPAFQG (SEQ ID NO: 165) rllmdy (SEQ ID NO: 10) adwa_VH_1.1 T28N+F29I (SEQ ID NO: 88) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) rllmdy (SEQ ID NO: 10) adwa_VH_1.2 T28N+F29I+R72A (SEQ ID NO: 89) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) rllmdy (SEQ ID NO: 10) adwa_VH_1.3 T28N+F29I+R72A+A49G+L79A (SEQ ID NO: 90) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) rllmdy (SEQ ID NO: 10) adwa_VH_1.4 T28N+F29I+R72A+N74T+A75S (SEQ ID NO: 91) dyymn (SEQ ID NO: 8) WIDPDNGNTIYDPKFQG (SEQ ID NO: 23) rllmdy (SEQ ID NO: 10) VH05-2(F64V) VK01 (SEQ ID NO: 93) dyymn (SEQ ID NO: 8) WIDPDQGNTIYEPKVQG (SEQ ID NO: 166) rllmdy (SEQ ID NO: 10)

Table 14 shows typical heavy chain CDRs according to Kabat.

Table 15: Typical light chain CDRs according to Kabat

VL (SEQ ID NO) CDR-L1 (SEQ ID NO) CDR-L2 (SEQ ID NO) CDR-L3 (SEQ ID NO) ADWA-11 VL mouse (SEQ ID NO: 21) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11 2.4 (SEQ ID NO: 7) rstkslshfngntylf (SEQ ID NO: 11) Yymsslas (SEQ ID NO: 12) qqsleypft (SEQ ID NO: 13) ADWA11_VK01 (1) (also referred to herein as adwa_VL_1.1 L46R)(SEQ ID NO: 47) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_1a (1) L29I (SEQ ID NO: 48) RSTKSILHFNGNTYLF (SEQ ID NO: 138) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_1b (1) L30S (SEQ ID NO: 49) rstkslshfngntylf (SEQ ID NO: 11) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_1c (1) T36S (SEQ ID NO: 50) RSTKSLLHFNGNSYLF (SEQ ID NO: 140) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_2a (1) Y55A (SEQ ID NO: 51) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YAMSNLAS (SEQ ID NO: 142) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_2b (1) M56A (SEQ ID NO: 52) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYASNLAS (SEQ ID NO: 144) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_2c (1) N58S (SEQ ID NO: 53) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) Yymsslas (SEQ ID NO: 12) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_2d (1) A60Q (SEQ ID NO: 54) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLQS (SEQ ID NO: 146) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_3a (1) M94Q (SEQ ID NO: 55) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) qqsleypft (SEQ ID NO: 13) ADWA11_VK01_3b (1) L97Y (SEQ ID NO: 56) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSYEYPFT (SEQ ID NO: 147) ADWA11_VK01_3c (1) E98S (SEQ ID NO: 57) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLSYPFT (SEQ ID NO: 149) ADWA11_VK01_3d (1) Y99T (SEQ ID NO: 58) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLETPFT (SEQ ID NO: 151) ADWA11_VK01_4a (1) F101L (SEQ ID NO: 59) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPLT (SEQ ID NO: 153) ADWA11_VK01_4b (1) F101W (SEQ ID NO: 60) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPWT (SEQ ID NO: 155) ADWA11_VK01_4c (1) Q105G (SEQ ID NO: 61) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11VK1 IGKV2-28 (SEQ ID NO: 62) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11VK2 IGKV2-30 (SEQ ID NO: 63) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11VK3 IGKV4-1 (SEQ ID NO: 64) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11VK4 IGKV1-39 (SEQ ID NO: 65) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11VK5 IGKV3-11 (SEQ ID NO: 66) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27) ADWA11_VK01_2.1 (SEQ ID NO: 67) rstkslshfngntylf (SEQ ID NO: 11) YYMSNLAS (SEQ ID NO: 26) qqsleypft (SEQ ID NO: 13) ADWA11_VK01_2.2 (SEQ ID NO: 68) rstkslshfngntylf (SEQ ID NO: 11) YYMSNLAS (SEQ ID NO: 26) MQSYEYPFT (SEQ ID NO: 147) ADWA11_VK01_2.3 (SEQ ID NO: 69) rstkslshfngntylf (SEQ ID NO: 11) YYASNLAS (SEQ ID NO: 144) qqsleypft (SEQ ID NO: 13) adwa_VL_1.2 L46R+Y36F (SEQ ID NO: 92) RSTKSLLHFNGNTYLF (SEQ ID NO: 25) YYMSNLAS (SEQ ID NO: 26) MQSLEYPFT (SEQ ID NO: 27)

Table 15 shows typical light chain CDRs according to Kabat.

Table 16: Typical Chotia Heavy Chain CDRs

VH (SEQ ID NO) CDR-H1 (SEQ ID NO) CDR-H2 (SEQ ID NO) CDR-H3 (SEQ ID NO) ADWA-11 VH mouse (SEQ ID NO: 20) GFNIKDYYMN (SEQ ID NO: 28) WIDPDNGN (SEQ ID NO: 29) RLLMDY (SEQ ID NO: 30) ADWA11 2.4 VH (SEQ ID NO: 6) gfnikdyymn (SEQ ID NO: 14) widpdqgn (SEQ ID NO: 15) rllmdy (SEQ ID NO: 16) ADWA11VH1 IGHV1-46(SEQ ID NO: 34) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH2 IGHV3-23 (SEQ ID NO: 35) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH3 IGHV3-30 (SEQ ID NO: 36) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH4 IGHV1-69(SEQ ID NO: 37) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH5 IGHV3-48 (SEQ ID NO: 38) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11 VH05_VK1 (also referred to herein as adwa_VH_1.5 T28N+F29I+R72A+A49G+L79A+N74T+A75S) (SEQ ID NO: 39) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH5 D61E (SEQ ID NO: 40) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH5 N55Q (SEQ ID NO: 41) gfnikdyymn (SEQ ID NO: 14) widpdqgn (SEQ ID NO: 15) rllmdy (SEQ ID NO: 16) ADWA11VH5 N28Q (SEQ ID NO: 42) gfnikdyymn (SEQ ID NO: 14) widpdqgn (SEQ ID NO: 15) rllmdy (SEQ ID NO: 16) ADWA11VH5K30A (SEQ ID NO: 43) GFNIADYYMN (SEQ ID NO: 159) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH5 N57Q (SEQ ID NO: 44) gfnikdyymn (SEQ ID NO: 14) WIDPDNGQ (SEQ ID NO: 164) rllmdy (SEQ ID NO: 16) ADWA11VH5 P62A (SEQ ID NO: 45) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) ADWA11VH5 K63A (SEQ ID NO: 46) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) adwa_VH_1.1 T28N+F29I (SEQ ID NO: 88) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) adwa_VH_1.2 T28N+F29I+R72A (SEQ ID NO: 89) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) adwa_VH_1.3 T28N+F29I+R72A+A49G+L79A (SEQ ID NO: 90) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) adwa_VH_1.4 T28N+F29I+R72A+N74T+A75S (SEQ ID NO: 91) gfnikdyymn (SEQ ID NO: 14) WIDPDNGN (SEQ ID NO: 29) rllmdy (SEQ ID NO: 16) VH05-2(F64V) VK01 (SEQ ID NO: 93) gfnikdyymn (SEQ ID NO: 14) widpdqgn (SEQ ID NO: 15) rllmdy (SEQ ID NO: 16)

Table 16 shows typical Chotia heavy chain CDRs.

Table 17: Typical Chotia light chain CDRs

VL (SEQ ID NO) CDR-L1 (SEQ ID NO) CDR-L2 (SEQ ID NO) CDR-L3 (SEQ ID NO) ADWA-11 VL mouse (SEQ ID NO: 21) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) QSLEYPFT (SEQ ID NO: 33) ADWA11 2.4 (SEQ ID NO: 7) STKSLSHFNGNTYL (SEQ ID NO: 17) yymss(SEQ ID NO: 18) qsleypft (SEQ ID NO: 19) ADWA11_VK01 (1) (also referred to herein as adwa_VL_1.1 L46R) (47) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_1a (1) L29I (SEQ ID NO: 48) STKSILHFNGNTYL (SEQ ID NO: 139) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_1b (1) L30S (SEQ ID NO: 49) STKSLSHFNGNTYL (SEQ ID NO: 17) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_1c (1) T36S (SEQ ID NO: 50) STKSLLHFNGNSYL (SEQ ID NO: 141) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_2a (1) Y55A (SEQ ID NO: 51) STKSLLHFNGNTYL (SEQ ID NO: 31) YAMSN (SEQ ID NO: 143) qsleypft (SEQ ID NO: 19) ADWA11_VK01_2b (1) M56A (SEQ ID NO: 52) STKSLLHFNGNTYL (SEQ ID NO: 31) YYASN (SEQ ID NO: 145) qsleypft (SEQ ID NO: 19) ADWA11_VK01_2c (1) N58S (SEQ ID NO: 53) STKSLLHFNGNTYL (SEQ ID NO: 31) yymss(SEQ ID NO: 18) qsleypft (SEQ ID NO: 19) ADWA11_VK01_2d (1) A60Q (SEQ ID NO: 54) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_3a (1) M94Q (SEQ ID NO: 55) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_3b (1) L97Y (SEQ ID NO: 56) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) QSYEYPFT (SEQ ID NO: 148) ADWA11_VK01_3c (1) E98S (SEQ ID NO: 57) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) QSLSYPFT (SEQ ID NO: 150) ADWA11_VK01_3d (1) Y99T (SEQ ID NO: 58) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) QSLETPFT (SEQ ID NO: 152) ADWA11_VK01_4a (1) F101L (SEQ ID NO: 59) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) QSLEYPLT (SEQ ID NO: 154) ADWA11_VK01_4b (1) F101W (SEQ ID NO: 60) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) QSLEYPWT (SEQ ID NO: 156) ADWA11_VK01_4c (1) Q105G (SEQ ID NO: 61) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11VK1 IGKV2-28 (SEQ ID NO: 62) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11VK2 IGKV2-30 (SEQ ID NO: 63) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11VK3 IGKV4-1 (SEQ ID NO: 64) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11VK4 IGKV1-39 (SEQ ID NO: 65) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11VK5 IGKV3-11 (SEQ ID NO: 66) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_2.1 (SEQ ID NO: 67) STKSLSHFNGNTYL (SEQ ID NO: 17) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19) ADWA11_VK01_2.2 (SEQ ID NO: 68) STKSLSHFNGNTYL (SEQ ID NO: 17) YYMSN (SEQ ID NO: 32) QSYEYPFT (SEQ ID NO: 148) ADWA11_VK01_2.3 (SEQ ID NO: 69) STKSLSHFNGNTYL (SEQ ID NO: 17) YYASN (SEQ ID NO: 145) qsleypft (SEQ ID NO: 19) adwa_VL_1.2 L46R+Y36F (SEQ ID NO: 92) STKSLLHFNGNTYL (SEQ ID NO: 31) YYMSN (SEQ ID NO: 32) qsleypft (SEQ ID NO: 19)

Table 17 shows typical Chotia light chain CDRs.

In some aspects, the antibody or antigen binding fragment thereof comprises CDR-L1, CDR-L2, and CDR-L3 as shown in the amino acid sequence of at least one of SEQ ID NO: 11-13.17-19, 25-27, 31-33, or 71 -76.

In some aspects, the antibody, or antigen binding fragment thereof, further comprises CDR-H1, CDR-H2, and CDR-H3 as indicated by the amino acid sequence of at least one of SEQ ID NOs: 8-10, 14-16, 22-24, or 28 -thirty.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 7, and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence SEQ ID NO:6.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 7 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 20.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 7 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence, any of SEQ ID NO: 34-46, 88-91 or 93.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 21 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as indicated by the amino acid sequence, any of SEQ ID NO: 47-69 or 92 and CDR-H1, CDR-H2 and CDR- H3 as shown in the amino acid sequence SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 5 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 2.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 5 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 3.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 5 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 124 or SEQ ID NO: 182.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 123 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 124 or SEQ ID NO: 182.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO: 123 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-L1, CDR-L2, and CDR-L3 as shown in the amino acid sequence encoded by the plasmid insert deposited with ATCC accession number PTA-124918.

In some aspects, the antibody, or antigen binding fragment thereof, comprises CDR-H1, CDR-H2, and CDR-H3 as shown in the amino acid sequence encoded by the plasmid insert deposited with ATCC accession number PTA-124917.

In some aspects, the antibody or antigen binding fragment thereof comprises CDR-L1, CDR-L2 and CDR-L3 in the amino acid sequence encoded by the plasmid insert deposited with ATCC accession number PTA-124918, and CDR-H1, CDR-H2 and CDR -H3 amino acid sequence encoded by the plasmid insert deposited at ATCC with accession number PTA-124917.

In some aspects, the antibody, or antigen binding fragment thereof, comprises a light chain variable region comprising an amino acid sequence encoded by a plasmid insert deposited with ATCC accession number PTA-124918.

In some aspects, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence encoded by a plasmid insert deposited with ATCC accession number PTA-124917.

In some aspects, the antibody or antigen binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:11, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:12, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:13, CDR-H1 containing the amino acid sequence of SEQ ID NO:8, CDR-H2 containing the amino acid sequence of SEQ ID NO:9 and CDR-H3 containing the amino acid sequence of SEQ ID NO:10.

In some aspects, the antibody or antigen binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:17, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:18, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:19, CDR-H1 containing the amino acid sequence of SEQ ID NO:14, CDR-H2 containing the amino acid sequence of SEQ ID NO:15 and CDR-H3 containing the amino acid sequence of SEQ ID NO:16.

In some aspects, the antibody, or antigen binding fragment thereof, may comprise a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOs: 6, 34-46, 88-91 and 93 (e.g., SEQ ID NOs : 6). VH may contain an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any of SEQ ID NO: 6, 34- 46, 88-91 and 93 (eg, SEQ ID NO: 6). VH may comprise an amino acid sequence of any of SEQ ID NO: 6, 34-46, 88-91 and 93 (eg, SEQ ID NO: 6).

In some aspects, the antibody, or an antigen binding fragment thereof, may comprise a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6. The VH may comprise an amino acid sequence that is at least 91%, 92%, 93% identical to that of SEQ ID NO: 6. 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6. VH may comprise the amino acid sequence of SEQ ID NO: 6. In some embodiments, an antibody or antigen binding fragment thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody, or an antigen binding fragment thereof, comprises a VH consisting of the amino acid sequence of SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding fragment thereof, may comprise a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NO: 7, 47-69 and 92 (eg, SEQ ID NO: 7). VL may contain an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any of SEQ ID NO: 7, 47- 69 and 92 (eg, SEQ ID NO: 7). VL may comprise an amino acid sequence of any of SEQ ID NO: 7, 47-69 and 92 (eg, SEQ ID NO: 7).

In some aspects, the antibody, or antigen binding fragment thereof, may comprise a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:7. VL may contain an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:7. VL may contain the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody or antigen binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or antigen binding fragment thereof comprises a VL consisting of the amino acid sequence SEQ ID NO: 7.

In some aspects, an antibody, or an antigen binding fragment thereof, may comprise a heavy chain comprising a VH comprising an amino acid sequence of any of SEQ ID NOs: 6, 34-46, 88-91 and 93 (e.g., SEQ ID NO: 6) and further comprising an IgG1 constant domain (eg, an IgG1 constant domain comprising the amino acid sequence of any of SEQ ID NO: 81, 82, 181 or 184). In some aspects, a variant antibody or antigen binding fragment comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions in the full-length heavy chain. In a further aspect, the embodiment has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least , 99% sequence identity with the full-length heavy chain, and wherein said antibody or antigen binding fragment specifically binds αvβ8 integrin.

In some embodiments, the antibody, or antigen binding fragment thereof, comprises a heavy chain comprising a VH containing the amino acid sequence of SEQ ID NO: 6 and further comprises an IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 184. In some in embodiments, the antibody, or antigen binding fragment thereof, comprises a VH consisting of the amino acid sequence of SEQ ID NO: 6 and further comprising an IgG1 constant domain consisting of the amino acid sequences of SEQ ID NO: 181 or SEQ ID NO: 184. In some embodiments, the antibody has no effector function(s). In further other embodiments, the antibody molecule has a heavy chain constant region selected from, for example , the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular selected from, for example , the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4 ( eg human). In another embodiment, the antibody molecule has a light chain constant region selected from , for example , a kappa light chain constant region (eg, encoded by the amino acid sequence SEQ ID NO: 83) or lambda ( eg , human). The constant region may be altered, e.g. , mutated, to modify the properties of the antibody ( eg , to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cellular function, and/or complement function). In one embodiment, the antibody has: an effector function; and can fix complement. In other embodiments, the antibody does not; attracts effector cells; or fixes complement. In another embodiment, the antibody has reduced or no ability to bind the Fc receptor. For example, there is an isotype or subtype, fragment, or other mutant that does not support Fc receptor binding, eg , it has a subtagenized or deleted Fc receptor binding region.

Methods for altering the constant region of an antibody are known in the art. Antibodies with altered function, for example altered affinity for an effector ligand such as an FcR on a cell, or a C1 complement component, can be prepared by replacing at least one amino acid residue in the constant region of the antibody with a different residue (see, for example, EP 388151 A1 , US Patent No. 5,624,821 and US Patent No. 5,648,260, all of which are incorporated herein by reference). A similar type of change may be described that, when applied to immunoglobulin from mice or other species, would reduce or eliminate these functions.

In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain comprising a VL comprising the amino acid sequence of any of SEQ ID NOs: 7, 47-69 and 92 (e.g., SEQ ID NO: 7) and further comprises a kappa constant domain containing amino acid sequence of SEQ ID NO: 83. In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain comprising a VL consisting of the amino acid sequence of SEQ ID NO: 7 and further comprises a kappa constant domain consisting of the amino acid sequence of SEQ ID NO: 83 .

In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain (HC) comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody or antigen binding fragment thereof comprises an HC comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody or antigen binding fragment thereof comprises an HC consisting of amino acid sequence SEQ ID NO: 2. In some embodiments, the antibody has no effector functions.

In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain (HC) comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or antigen binding fragment thereof comprises an HC comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody or antigen binding fragment thereof comprises an HC consisting of the amino acid sequence SEQ ID NO: 3. In some embodiments, the antibody has no effector functions. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain (LC) comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to SEQ ID NO: 5. In some embodiments, the antibody or antigen binding fragment thereof comprises an LC comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen binding fragment thereof comprises an LC consisting of the amino acid sequence SEQ ID NO: 5. NO: 5. In some embodiments, the antibody does not have an effector function.

Germline replacements

In certain embodiments, the antibody or antigen binding fragment thereof comprises the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO:22, CDR-H2 containing SEQ ID NO:23 and CDR-H3 containing SEQ ID NO:24 ; and/or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO:25 or 71, CDR-L2 containing SEQ ID NO:26 or 72 and CDR-L3 containing SEQ ID NO:27 or 73. In certain in embodiments, the antibody or antigen binding fragment thereof comprises the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO:28, CDR-H2 containing SEQ ID NO:29 and CDR-H3 containing SEQ ID NO:30; and/or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO:31 or 74, CDR-L2 containing SEQ ID NO:32 or 75 and CDR-L3 containing SEQ ID NO:33 or 76. They are The mouse and VL CDRs are preferably grafted or otherwise added in the context of the human VH and VL domain. A variety of human germline acceptor sequences are available, and the method of “humanizing” an antibody from a non-human species for use in humans is well known in the art and is also discussed elsewhere herein. Therefore, one skilled in the art will appreciate that the above mouse CDR sequences can be placed in the context of human V domain amino acid sequences. This typically involves changes to the human germline acceptor sequences to retain antibody binding and other desirable characteristics of the original parent (ie, donor) antibody. Both CDR and frame regions (FW) can be constructed as follows.

In certain embodiments, no more than 11 or no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 substitution is made in CDR-L1, relative to amino acid sequence SEQ ID NO: 25, 31, 71 or 74. In certain embodiments, no more than 6, no more than 5, no more than 4 , no more than 3, no more than 3, no more than 2, or no more than one substitution is made in CDR-L2, relative to the amino acid sequence of SEQ ID NO: 26, 32, 72, or 75. In certain embodiments, no more than 8, not more than 7, not more than 6, not more than 5, not more than 4, not more than 3, not more than 3, not more than 2 or not more than one substitution made in CDR-L3, relative to the amino acid sequences SEQ ID NO: 27, 33, 73, 76. In some embodiments, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in CDR-H1, relative to the amino acid sequence of SEQ ID NO: 22 or 28. In some embodiments, no more than no more than 17, no more no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11 or no more than 10, no more than 9, no more than 8, no more than 7, no more more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in CDR-H2 relative to the amino acid sequence of SEQ ID NO: 23 or 29. In some embodiments, no more than 12, no more than 11 or no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 substitution is made in CDR-H3, relative to the amino acid sequence of SEQ ID NO: 24 or 30. In certain embodiments, the substitution(s) do not change the binding affinity value (K _D ) by more than 1000-fold , more than 100-fold or 10-fold. In certain embodiments, the substitution is a conservative substitution according to Table 1.

In certain embodiments, the substitution is a human germline substitution in which the (donor) CDR residue is replaced with a corresponding human germline (acceptor) residue to increase the human amino acid content and potentially reduce the immunogenicity of the antibody, as described in, for example, US patent application publication No. 2017/0073395 and Townsend et al., 2015, Proc. Nat. Acad. Sci. USA 112(50):15354-15359).

Methods and libraries for introducing human germline residues into antibody CDRs are described in detail in US Patent Application Publication No. 2017/0073395 and Townsend et al., 2015, Proc. Natl. Acad. Sci. USA. 112(50):15354-15359, and both documents are incorporated herein by reference in their entirety.

The antibody or antigen binding fragment thereof may comprise a VH framework region comprising a human germline VH framework sequence. The VH framework sequence may be from human germline VH3, germline VH1, germline VH5, or germline VH4. Preferred human germline heavy chain framework regions are those derived from the VH1, VH3 or VH5 germline. For example, VH framework regions of the following germline type may be used: IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48 (germline names are based on the IMGT definition of germline). Preferred human germline light chain framework regions are those derived from the Vκ or Vλ germline. For example, VL framework regions of the following germline type may be used: IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11 (germline names are based on the IMGT definition of germline). Alternatively or in addition, the framework sequence may be a human germline consensus framework, such as the Vλ1 human consensus framework, Vκ1 consensus, Vκ2 consensus, Vκ3 consensus, VH3 germline consensus, VH1 germline consensus, VH5 germline consensus sequence or VH4 germline consensus sequence. Human germline framework sequences are available from various public databases such as V-base, IMGT, NCBI or Abysis.

The antibody or antigen binding fragment thereof may comprise a VL framework region comprising a human germline VL framework sequence. The VL framework region may contain one or more amino acid substitutions, additions or deletions while still maintaining functional and structural similarity to the germline from which it is derived. In some aspects, the VL frame region is at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97% at least at least 98%, at least 99% or 100% identical to the human germline VL framework sequence. In some aspects, the antibody or antigen binding fragment thereof comprises a VL framework region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions, or deletions relative to a human germline VL framework sequence. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions are in the framework regions only. In some aspects, % identity is based on similarity to the VL except for those portions herein defined as CDRs.

The human germline VL framework may be the DPK9 framework (IMGT name: IGKV1-39, eg SEQ ID NO:128). The human germline VL framework may be the IGKV2-28 framework. The human germline VL framework region may be the DPK18 framework region (IMGT name: IGKV2-30). The human germline VL framework region may be the DPK24 framework region (IMGT name: IGKV4-1). The human germline VL framework region may be the HK102_V1 framework region (IMGT name: IGKV1-5). The human germline VL framework region may be the Vg_38K framework region (IMGT name: IGKV3-11). The human germline VL framework may be a human Vλ consensus framework. The human germline VL framework region may be the human Vλ1 framework consensus sequence. The human germline VL framework may be the human Vλ3 framework consensus sequence. The human germline VL framework may be a human Vκ consensus framework. The human germline VL framework may be the human Vκ1 framework consensus sequence. The human germline VL framework may be the human Vκ2 framework consensus sequence. The human germline VL framework may be the human Vκ3 framework consensus sequence.

In some aspects, the VL framework region is DPK9 (SEQ ID NO: 128). Other similar framework regions are also predicted to deliver advantageous antibodies of the invention comprising the CDRs of SEQ ID NOs: 11-13 and 17-19; and CDRs characterized by the following VL amino acid sequences: 7, 47-69 and 92, including, for example, IGKV2-28, IGKV2-30, IGKV4-1 or IGKV3-11, which may have 99, 97, 97, 96, 80, 76, 66, 97, 97, 96, 76 and 74% identity, respectively, to the FW region of DPK-9 and one or more amino acid differences in common structural (Kabat numbering) (A) residues immediately downstream of the CDR (Vernier region), L2, L4, L35, L36, L46, L47, L48, L49, L64, L66, L68, L69, L71, (B) VH/VL chain packing residues: L36, L38, L44, L46, L87 and (C) canonical CDR scaffold residues L2, L48, L64, L71 (see Lo, "Antibody Humanization by CDR Grafting", (2004) Antibody Engineering, Vol. 248, Methods in Molecular Biology pp 135-159 and O'Brien and Jones, "Humanization of Monoclonal Antibodies by CDR Grafting", (2003) Recombinant Antibodies for Cancer Therapy, Vol. 207, Methods in Molecular Biology pp 81-100). Particularly preferred are the framework regions of IGKV2-28, IGKV2-30, IGKV4-1 or IGKV3-11, having 99, 97, 97, 96, 80, 76, 66% identity with DPK9, respectively, and having no amino acid differences in these common structural features. In some aspects, % identity is based on similarity to the VL, excluding those portions herein defined as CDR.

The antibody or antigen binding fragment thereof may comprise a VH framework region comprising a human germline VH framework sequence. The VH framework region may contain one or more amino acid substitutions, additions or deletions while still maintaining functional and structural similarity to the germline from which they are derived. In some aspects, the VH framework sequence has at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97% at least at least 98%, at least 99% or 100% identity to the human germline VH framework sequence. In some aspects, the antibody or antigen binding fragment thereof comprises a VH framework sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions or deletions relative to the human germline VH framework sequence. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions are in the framework regions only. In some aspects, % identity is based on similarity to VH excluding those portions herein defined as CDR.

The human germline VH framework sequence may be, for example, the framework region of IGHV3-07 (also known as DP-54), IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48. The human germline VH framework sequence may be the VH framework region of a human germline consensus sequence. The human germline VH framework sequence may be the human germline consensus sequence VH3 framework region. The human germline VH framework sequence may be the human germline consensus sequence VH5 framework region. The human germline VH framework sequence may be the VH1 framework region of a human germline consensus sequence. The human germline VH framework sequence may be the human germline consensus sequence VH4 framework region.

In some aspects, the VH framework sequence is IGHV3-07 (SEQ ID NO: 127). Other similar framework regions are also predicted to deliver advantageous antibodies of the invention comprising the CDRs of SEQ ID NOs: 8-10 and 14-16, and the CDRs characterized by the following VH amino acid sequences: SEQ ID NOs: 6, 34-46, 88- 91 and 93, including IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69 or IGHV3-48, which may have 93, 92, 92, 99, 97, 97, 96, 96, 94, 94, 93, 92% identity, respectively, to the FW region of DP-54, and one or more amino acid differences in general structural features (numbering according to Kabat) (A) residues immediately downstream of the CDR (Vernier region), H2, H47, H48 and H49, H67, H69, H71, H73, H93, H94, (B) VH/VL chain packing residues: H37, H39, H45, H47, H91, H93 and (C) canonical CDR scaffold residues H24, H71, H94 (see Lo 2004 and O'Brien and Jones 2003). The typical framework regions of DP-50, IGHV3-30*09, IGHV3-30*15 have 93, 92 and 92% identity with DP-54, respectively, and have no amino acid differences in these common structural features. In some aspects, % identity is based on similarity to VH, excluding those portions herein defined as CDR.

In certain embodiments, an antibody or antigen binding fragment thereof described herein comprises (i) a VH comprising an amino acid sequence that is at least 50% at least 60% at least 70% at least 75% at least at least 80% at least 85% at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97% at least at least 98% at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:6, and/or (ii) VL containing an amino acid sequence that is at least 50% at least 60% at least 66% identical to at least 70% at least 75% at least 76% at least 80% at least 85% at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97% at least 98% at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:7. Any combination of these VL and VH sequences is also covered by the invention.

In certain embodiments, an antibody or antigen binding fragment thereof described herein comprises (i) an HC comprising an amino acid sequence that is at least 50% at least 60% at least 70% at least 75% at least at least 80% at least 85% at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97% at least at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3; and/or (ii) an LC containing an amino acid sequence that is at least 50% at least 60% at least 70% at least 75% at least 80% at least 85% at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97% at least 98% at least 99% or 100% identical in amino acid sequence SEQ ID NO:5. Any combination of these HC and LC sequences is also covered by the invention.

In certain embodiments, an antibody or antigen binding fragment thereof described herein comprises an Fc domain. The Fc domain may be derived from IgA (eg IgA ₁ or IgA ₂ ), IgG, IgE or IgG (eg IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ).

The invention further provides an antibody or antigen binding fragment thereof that competes for binding to human αvβ8 integrin latency associated peptide (LAP). For example, if binding of an antibody or an antigen binding fragment thereof to a human αvβ8 integrin interferes with the subsequent binding of LAP to a human αvβ8 integrin, then the antibody or antigen binding fragment thereof competes with LAP for binding of the human αvβ8 integrin.

Antibodies and antigen binding fragments provided by the invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab', F(ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), their mutants, fusion proteins containing an antibody fragment, an antibody domain (dAb), humanized antibodies and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants and covalently modified antibodies. Antibodies and antigen-binding fragments can be derived from mouse, rat, human, or any other species (including chimeric or humanized antibodies). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody.

Biological activity of anti- α v β 8 integrin antibodies

In addition to binding an epitope on αvβ8 integrin, an antibody or antigen binding fragment thereof of the invention may mediate biological activity. That is, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin and mediates at least one detectable activity selected from the following:

(i) binds specifically to αvβ8 integrin (eg, human, mouse, cynomolgus and/or rat αvβ8 integrin);

(ii) reduces the interaction between αvβ8 integrin and latency-associated peptide (LAP);

(iii) reduces TGF-β signaling;

(iv) effectively blocks αvβ8 integrin-mediated activation of TGFβ with an IC50 ≤10 nM;

(v) has a comparable Kd (within 5-fold) to the non-human primate (NHP) ortholog;

(vi) selectively binds human αvβ8 and does not detectably bind αvβ8 homolog (eg, αvββ1, αvβ3, αvβ5 and αvβ6);

(vii) causes suppression of growth and/or complete tumor regression in an animal model of cancer selected from squamous cell carcinoma, breast cancer and colon cancer, alone or in combination with an immunomodulator, for example, a modulator of checkpoint inhibitors, for example, PD-1 inhibitors, PD-L1, CTLA-4 or stimulatory molecule agonist, 4-1BB;

(viii) causes suppression of tumor growth and/or complete tumor regression in an animal model of cancer in combination with anticancer therapy, such as radiotherapy;

(ix) demonstrates at least a 60% reduction in tumor growth in a syngeneic xenograft tumor model, for example, when administered at a dose of ≤10 mg/kg;

(x) enhances the antitumor response in the presence of one or more immunomodulators, for example, a checkpoint inhibitor antagonist or a checkpoint activator agonist, for example, a PD-1, PD-L1 or CTLA-4 antagonist or an immune response activator, for example, a 4-1BB agonist , when administered to a subject, for example, a mouse or a human;

(xi) has an efficacy that is independent of αvβ8 integrin expression in a tumor model;

(xii) increases the number of CD8+ GzmB+ T cells in the tumor microenvironment, for example, as monotherapy;

(xiii) shows a reduction, e.g., at least >80% reduction in tumor growth when used in combination with a checkpoint inhibitor antagonist (e.g., anti-PD-1 or anti-PD-L1 antibody), e.g., in a syngeneic model of squamous cell carcinoma, breast cancer and/or colon cancer;

(xiv) shows a statistically significant improvement in overall survival in the subject, e.g., human or mouse, as determined by Kaplan-Meier analysis;

(xv) exhibits suitable formulation properties, including a high degree of thermal stability and minimal aggregation at high concentrations; or

(xvi) can show reproducible expression and purity under industrial production conditions.

In some embodiments, the anti-αvβ8 integrin antibody or antigen binding fragment thereof has at least one of the following properties:

i. binding affinity, expressed as KD, for human αvβ8 integrin that is less than the KD of mouse ADWA11 antibody, e.g., less than 536 pM (e.g., 1.5, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 150, 200, 250, 300, 350, 370, 400, 450, 500, 510, 520, 530, 531, 532, 533, 534 or 535 pM);

ii. KD for human αvβ8 integrin that is less than or equal to 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 , 90 or 100 pM), for example, for purified human αvβ8 integrin;

iii. KD for mouse αvβ8 integrin that is less than the KD of mouse ADWA11 antibody, e.g., less than 489 pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 , 250, 300, 350, 370, 400, 450, 460, 470, 480, 485, 486, 487 or 488 pM);

iv. KD for cynomolgus αvβ8 integrin that is less than the KD of mouse ADWA11 antibody, e.g., less than 507 pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 500, 501, 502, 503, 504, 505 or 506 pM);

v. KD for rat αvβ8 integrin, which is approximately 160 pM;

vi. shows approximately equivalent affinities for at least two, three, or all human, cynomolgus, mouse, and rat αvβ8 integrins, e.g., with a KD that is less than 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 pM), for example, determined using the Biacore affinity assay;

vii. IC50 for inhibition of TGFβ transactivation that is less than the mouse ADWA11 antibody, e.g., less than 183 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 181 or 182 pM);

viii. IC50 for inhibition of TGFβ transactivation in U251 cells is approximately 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320 or 340 pM;

ix. EC50 for U251 cells is approximately 126 pM with a standard deviation of plus/minus 34 pM;

X. EC50 for U251 cells is approximately 256 pM with a standard deviation of plus/minus 115 pM;

xi. EC50 for C8-S cells is approximately 115 pM;

xii. EC50 for C8-S cells is approximately 145 pM with a standard deviation of plus/minus 23.7 pM;

xii. at least one predicted human pharmacokinetic (PK) parameter selected from:

A. clearance from the central chamber (CL) is approximately 0.12-0.15 ml/h/kg;

b. intercompartmental clearance distribution (CLF) approximately 0.15-0.51 ml/h/kg;

With. volume of distribution for the central chamber (V1) approximately 36-39 ml/kg;

d. volume of distribution for the peripheral chamber (V2) approximately 21-33 ml/kg; and/or

e. the final half-life (t _1/2 ) is approximately 12-17 days; or

xiv. does not show detectable binding to human Fcγ receptors or C1q.

In some embodiments, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof binds αvβ8 integrin+ cells (eg, cells expressing αvβ8 integrin) with high apparent affinity. Apparent binding affinity can be assessed using flow cytometry to determine the binding of the antibody to cells expressing the target protein (eg, αvβ8 integrin). Cells can be transiently or stably transfected with a nucleic acid encoding the αvβ8 integrin. Alternatively, the cells may be cells that naturally express αvβ8 integrin on their surface. Regardless of the sources of αvβ8 integrin+ cells, antibody binding to cells can be readily assessed using a variety of methods known in the art. The antibody or antigen-binding fragment thereof binds human αvβ8 integrin, cynomolgus αvβ8 integrin, mouse αvβ8 integrin, and rat αvβ8 integrin.

The invention includes an antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin and antagonizes αvβ8 integrin-mediated activities (eg, TGFβ signaling, which may be mediated by inhibition of αvβ8 integrin interaction with latency-associated peptide (LAP)). A variety of assays are known in the art to determine inhibition of activity mediated by TGFβ signaling. One such assay is the TGFβ pathway transactivation assay. In one example of such an assay, the expression of SMAD, which can serve as a marker of TGFβ signaling and pathway activation, is monitored using a luciferase reporter. The ability of an anti-αvβ8 integrin antibody to bind αvβ8 integrin and antagonize the action of TGFβ signaling (eg, by inhibiting the interaction of αvβ8 integrin with LAP) is thus assessed by measuring the level of SMAD expressed in the presence or absence of the antibody. Preferably, the antibody may mediate a dose-dependent reduction in luciferase with an EC50 of about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 , pM, about 225 pM, about 250 pM, about 275 pM, about 300 pM, about 400 pM, or about 500 pM (e.g., EC50 between about 1 pM and about 100 pM, e.g., EC50 between about 1 pM and about 200 pM e.g., EC50 between about 1 pM and about 300 pM, e.g., EC50 between about 1 pM and about 400 pM, e.g., EC50 between about 1 pM and about 500 pM). More preferably, the antibody or antigen binding fragment thereof inhibits TGFβ signaling (e.g., TGFβ transactivation, e.g., TGFβ transactivation of SMAD) with an EC50 of about 100 pM (e.g., an EC50 between about 5 pM and about 175 pM, e.g., an EC50 between about 25 pM and about 175 pM, e.g. EC50 about 100 pM, about 105 pM, about 110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about 135 pM, about 140 pM, about 145 pM, about 150 pM , about 155 pM, about 160 pM, about 165 pM, about 170 pM, or about 175 pM). In some embodiments, the antibody or antigen binding fragment thereof inhibits TGFβ signaling (e.g., TGFβ transactivation, e.g., TGFβ SMAD transactivation) with an EC50 of less than about 5 nM (e.g., less than about 0.001, 0.01, 0.1, 0 ,2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4 , 4.5, 5, 5.1, 5.5, 6, 7, 8, 9, 10, 15, 20 or 25 nM). In embodiments, the antibody or antigen binding fragment thereof inhibits TGFβ signaling (e.g., TGFβ transactivation, e.g., TGFβ SMAD transactivation) with an EC50 of about 5 nM (e.g., about 0.001, 0.01, 0.1, 0.2, 0. 3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.1, 5.5, 6, 7, 8, 9, 10, 15, 20 or 25 nM).

In some embodiments, the antibody or antigen binding fragment thereof of the present disclosure inhibits TGFβ signaling with an EC50 of approximately 145 +/- 23.7 pM. In some embodiments, the antibody or antigen binding fragment thereof of the present disclosure inhibits TGFβ signaling at C8-S with an EC50 of approximately 145 +/- 23.7 pM. In some embodiments, the antibody or antigen binding fragment thereof of the present disclosure inhibits TGFβ signaling in C8-S cells with an EC50 of about 110 pM to about 180 pM.

In some embodiments, the antibody or antigen binding fragment thereof of the present disclosure inhibits TGFβ signaling with an EC50 of approximately 256 +/- 115 pM. In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure inhibits TGFβ signaling in U251 cells with an EC50 of approximately 256 +/- 115 pM. In some embodiments, the antibody or antigen binding fragment thereof of the present disclosure inhibits TGFβ signaling in U251 cells with an EC50 of about 80 pM to about 400 pM.

The invention covers an antibody or antigen-binding fragment thereof that binds human αvβ8 integrin, but does not detectably bind human αvβ3 integrin, αvβ5 integrin or αvβ6 integrin proteins.

III. EXPRESSION AND PRODUCTION OF ANTI- α v β 8 INTEGRIN ANTIBODIES

Nucleic acids encoding anti- α v β 8 integrin antibodies

The invention also provides polynucleotides encoding any antibodies, including antibody fragments and modified antibodies described herein. The invention also provides a method for producing any of the polynucleotides described herein. Polynucleotides can be produced and expressed by methods known in the art.

The sequence of the desired antibody, a specific antibody fragment or antigen-binding fragment thereof, and a nucleic acid encoding said antibody or fragment thereof can be determined using standard sequencing techniques. A nucleic acid sequence encoding a desired antibody, a specific antibody fragment, or an antigen binding fragment thereof can be inserted into various vectors (such as cloning and expression vectors) for recombinant production and characterization. A nucleic acid encoding a heavy chain, a specific antibody fragment, or a heavy chain antigen binding fragment, and a nucleic acid encoding a light chain, a specific antibody fragment, or a light chain antigen binding fragment, may be cloned into the same vector or different vectors.

The polynucleotide encoding the amino acid sequences above encodes an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and more preferably, identical to the amino acid sequence of the antibody or antigen binding fragment thereof of the present invention, as described herein.

In some embodiments, the invention provides polynucleotides encoding one or more proteins containing an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 3, and 5-76 (for example, a polynucleotide containing the sequence specified in SEQ ID NO: 1, 4, 183, 185, 186, 189, 190 or 191) or a nucleotide sequence encoding an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and, more preferably, identical to SEQ ID NO: 2, 3, 5-76, 88-93, 123, 124 and 182.

The invention provides polynucleotides containing a nucleic acid sequence represented in one or more of SEQ ID NO: 1, 183, 189 or 191 and encoding an antibody heavy chain or nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or identical to SEQ ID NO: 1, 183, 189 or 191 and heavy chain encoding antibodies.

The invention provides polynucleotides containing a nucleic acid sequence represented in one or more of SEQ ID NO: 4 or 185 and encoding an antibody light chain or nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or identical to SEQ ID NO: 4 or 185 and encoding an antibody light chain.

The invention provides polynucleotides containing the nucleic acid sequence set forth in SEQ ID NO: 190 and encoding the antibody heavy chain variable region or nucleotide sequence in at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% or identical to SEQ ID NO: 190 and encoding the variable region of the antibody heavy chain.

The invention provides polynucleotides containing the nucleic acid sequence set forth in SEQ ID NO: 186 and encoding at least 80%, 85%, 90%, 91%, 92%, 93% of the antibody light chain variable region or nucleotide sequence , 94%, 95%, 96%, 97%, 98%, or 99%, or identical to SEQ ID NO: 186 and encoding an antibody light chain variable region.

The invention provides polynucleotides containing the nucleic acid sequence set forth in SEQ ID NO: 192 or 193 and encoding the antibody heavy chain constant region or nucleotide sequence in at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99%, or identical to SEQ ID NO: 192 or 193, and encoding the antibody heavy chain constant region.

The invention provides polynucleotides containing the nucleic acid sequence set forth in SEQ ID NO: 194 and encoding an antibody light chain constant region or nucleotide sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% or identical to SEQ ID NO: 194 and encoding the antibody light chain constant region.

The invention provides a polynucleotide containing the nucleic acid sequence set forth in SEQ ID NO:189. The invention provides a polynucleotide containing the nucleic acid sequence set forth in SEQ ID NO: 190. The invention provides a polynucleotide containing the nucleic acid sequence set forth in SEQ ID NO: 185.

The invention provides a polynucleotide containing one or both nucleic acid sequences of the DNA insert of a plasmid deposited with ATCC and having Accession No. PTA-124917 and/or Accession No. PTA-124918.

The invention provides a polynucleotide containing the nucleic acid sequence of the insert in a plasmid deposited with ATCC and having Accession No. PTA-124917, and/or Accession No. PTA-124918.

The invention provides cells containing one or more nucleic acid molecules shown in SEQ ID NO: 1, 183 and 4.

The invention provides cells containing one or more nucleic acid molecules shown in SEQ ID NO: 185, 189 and 190.

In another aspect, the invention provides polynucleotides and variants thereof encoding an anti-αvβ8 integrin antibody (e.g., a human anti-αvβ8 integrin antibody), wherein such polynucleotide variants have at least 70%, at least 75%, at least 80% of at least 85% at least 87% at least 89% at least 90% at least 91% at least 92% at least 93% at least 94% at least 95% at least 96% at least 97%, at least 98%, or at least 99% sequence identity with any of the defined nucleic acid sequences described herein. These amounts are not intended to be limiting, and steps between the percentages stated are specifically contemplated as part hereof.

The invention provides polypeptides encoded by the nucleic acid molecules described herein.

In one embodiment, the VH and VL domains, or antigen binding fragments thereof, or full-length HC or LC, are encoded by separate polynucleotides. Alternatively, both VH and VL, or an antigen binding fragment thereof, or HC and LC, are encoded by a single polynucleotide.

Polynucleotides complementary to any such sequences are also covered by the present description. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include hnRNA molecules, which contain introns and correspond one-to-one to the DNA molecule, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but not necessarily, be present in the polynucleotide of the present disclosure, and the polynucleotide may, but not necessarily, be associated with other molecules and/or support materials.

Polynucleotides may contain a native sequence (ie, an endogenous sequence that encodes an antibody or fragment thereof) or may contain a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not impaired relative to the native immunoreactive molecule. The effect on the immunological reactivity of the encoded polypeptide can generally be assessed as described herein. In some embodiments, the variants exhibit at least about 70% identity, in some embodiments at least about 80% identity, in some embodiments at least about 90% identity, and in some embodiments at least about 95% identity to a polynucleotide sequence that encodes a native antibody or a fragment thereof. These amounts are not intended to be limiting, and steps between the percentages stated are specifically intended to be part of this document.

Two polynucleotide or polypeptide sequences are said to be “identical” if the nucleotide or amino acid sequences in the two sequences are the same when aligned for maximum matching, as described below. Comparisons between two sequences are typically made by comparing sequences in a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein refers to a segment of at least about 20 contiguous positions, typically 30 to about 75 or 40 to about 50, where the sequence can be compared to a reference sequence with the same number of contiguous positions after optimal alignment of two sequences.

Optimal sequence alignment for comparison can be performed using the MegAlign® program in the Lasergene® bioinformatics software package (DNASTAR®, Inc., Madison, WI), using default parameters. This program includes several alignment schemes, described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D. G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

In some embodiments, "percentage sequence identity" is determined by comparing two optimally aligned sequences in a comparison window of at least 20 positions, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) in the amount 20 percent or less, typically 5 to 15 percent or 10 to 12 percent, compared to reference sequences (which do not contain additions or deletions) for optimal alignment of two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues exist in both sequences, yielding the number of matching positions, and dividing the number of matching positions by the total number of positions in the reference sequence (i.e., window size) and multiplying results to 100 to obtain the percentage of sequence identity.

Variants may also or alternatively be substantially homologous to the native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions with the natural DNA sequence encoding the native antibody (or complementary sequence).

Suitable “moderately stringent conditions” include pre-wash in 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C-65°C, 5X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

As used herein, “very stringent conditions” or “high stringency conditions” include those where: (1) low ionic strength and high temperature are used for washing, for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0. 1% sodium dodecyl sulfate at 50°C; (2) a denaturing agent such as formamide is used during hybridization, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium -phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated sperm DNA salmon (50 µg/ml), 0.1% SDS and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2X SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1X SSC containing EDTA at 55°C. One skilled in the art will understand how to adjust temperature, ionic strength, etc. if necessary, to accommodate factors such as probe length and the like.

Those skilled in the art will appreciate that, as a result of the degeneration of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. However, polynucleotides that differ due to differences in codon usage are specifically contemplated herein. In addition, alleles of genes containing polynucleotide sequences presented herein are included within the scope of this description. Alleles are endogenous genes that are altered by one or more mutations or changes, such as deletions, additions and/or nucleotide substitutions. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard methods (such as hybridization, amplification and/or comparison of sequence databases).

The polynucleotides of the present description can be obtained using chemical synthesis, recombinant methods or PCR. Methods for chemically synthesizing polynucleotides are well known in the art and do not need to be described in detail herein. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to obtain the desired DNA sequence.

To produce polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector, in turn, can be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells by any methods known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-pairing or electroporation. Once introduced, the exogenous polynucleotide may be retained in the cell as an unintegrated vector (such as a plasmid) or integrated into the genome of the host cell. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al . , 1989.

Alternatively, PCR allows DNA sequences to be reproduced. PCR technology is well known in the art and is described in US Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al . eds., Birkauswer Press, Boston, 1994.

The RNA can be produced by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. Once the cell has replicated and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, as presented in Sambrook et al . , 1989, for example.

In some embodiments, the first vector contains a polynucleotide encoding a heavy chain and the second vector contains a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in equal amounts (such as equal molar amounts or equal mass amounts). In some embodiments, a molar or weight ratio of from 5:1 to 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a weight ratio of 1:1 to 1:5 is used for the heavy chain encoding vector and the light chain encoding vector. In some embodiments, a 1:2 weight ratio is used for the heavy chain encoding vector and the light chain encoding vector.

Vectors

In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells or NSO cells. Examples of such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

Suitable cloning and expression vectors may include a variety of components, such as a promoter, enhancer, and other transcriptional control sequences. The vector can also be designed to allow subsequent cloning of the antibody variable domain into various vectors. Suitable cloning vectors can be constructed according to standard methods, or can be selected from the large number of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell to be used, useful cloning vectors will generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and/or may carry marker genes that can be used when selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, for example pUC18, pUC19, Bluescript (eg pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vector such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen. Additionally, expression vectors are presented. Expression vectors are typically replicable polynucleotide constructs that contain a polynucleotide as described. It is understood that the expression vector must replicate in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids and expression vector(s) disclosed in PCT Publication No. WO 87/04462. Vector components may typically include, but are not limited to, one or more of the following: a signal sequence; origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers and terminator). Expression (i.e., translation) also typically requires one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

Vectors containing polynucleotides of interest and/or the polynucleotides themselves can be introduced into a host cell by any of a variety of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microparticle bombardment; lipofection; and infection (eg, where the vector is an infectious agent such as vaccinia virus). The choice of vectors or polynucleotides to be introduced often depends on the characteristics of the host cell.

Host cells

The antibody or antigen binding fragment thereof can be produced recombinantly using a suitable host cell. A nucleic acid encoding an antibody or an antigen-binding fragment thereof can be cloned into an expression vector, which can then be introduced into a host cell, such as an E. coli cell, a yeast cell, an insect cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell ) or a myeloma cell, where the cell does not otherwise produce immunoglobulin protein, to produce antibody synthesis in a recombinant host cell. Preferred host cells include a CHO cell, a human embryonic kidney HEK-293 cell, or an Sp2.0 cell, among many cells well known in the art. The antibody fragment can be obtained by proteolytic or other degradation of the full-length antibody, recombinant methods, or chemical synthesis. The polypeptide fragment of an antibody, especially shorter polypeptides up to about 50 amino acids, is conveniently prepared by chemical synthesis. Methods for the chemical synthesis of proteins and peptides are known in the art and are commercially available.

In various embodiments, anti-αvβ8 integrin heavy chains and/or anti-αvβ8 integrin light chains can be expressed in prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells (such as droddy), plant cells, insect cells and mammalian cells. Such expression can be carried out, for example, according to methods known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-αvβ8 integrin heavy chains and/or anti-αvβ8 integrin light chains may be expressed in yeast. See, for example, US Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications of anti-αvβ8 integrin heavy chains and/or anti-αvβ8 integrin light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into the desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells according to any suitable method.

Anti-αvβ8 integrin antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind the constant regions of the antibody. For example, a protein A, protein G, protein A/G, or antibody affinity column can be used to bind the constant region and purify an anti-αvβ8 integrin antibody. Hydrophobic interaction chromatography, such as a butyl or phenyl column, may also be suitable for the purification of some polypeptides. Many methods for purifying polypeptides are known in the art.

In some embodiments, the anti-αvβ8 integrin antibody is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol.Adv. 21: 695-713 (2003).

IV. USES AND MEDICAL THERAPIES

Therapeutic Use

In some aspects, the invention provides therapeutic methods for inhibiting αvβ8 integrin activity using an anti-αvβ8 integrin antibody or an antigen binding fragment thereof, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-αvβ8 integrin antibody or an antigen binding fragment thereof. A disorder to be treated is any disease or condition that is improved, ameliorated, inhibited, or prevented by removing, inhibiting, or reducing αvβ8 integrin activity or signaling. In particular, the anti-αvβ8 integrin antibodies of the invention, including humanized and chimeric antibodies, can be used for the prevention, treatment and/or amelioration of diseases, disorders or conditions caused by and/or associated with aberrant (e.g., increased) αvβ8 integrin activity and/or or TGFβ signaling in a subject (eg, in the tumor microenvironment of a subject suffering from cancer). In some embodiments, the disease, disorder, or condition includes a respiratory condition (eg, asthma), fibrosis, or anemia. In some embodiments, the disease, disorder, or condition is treatable or preventable with a vaccine.

In some aspects, the invention provides a method for selectively inhibiting αvβ8-dependent activation of latent-TGFβ in cells of the tumor microenvironment, including, for example, dendritic cells, T-regulatory cells, tumor-associated macrophages, and/or cells of the tumor itself. Without being bound by any particular theory, more precise and selective antagonism of TGFβ activation in the immune system and/or tumor microenvironment mediated by administration of an anti-αvβ8 integrin antibody of the invention may promote an antitumor immune response in a subject without disrupting the broader homeostatic functions of TGFβ. An antitumor immune response by a subject that does not disrupt the broader homeostatic functions of TGFβ can be expected to provide safety and therapeutic benefits over systemic TGFβ inhibition.

The methods provided herein can also be used to reduce and/or attenuate TGFβ activity (eg, TGFβ tumor-promoting activity) in a subject (eg, within the tumor microenvironment of a subject suffering from cancer). TGFβ activity affecting angiogenesis, metastasis, epithelial-mesenchymal transition, and/or suppression of infiltrating immune cells (e.g., tumor-infiltrating lymphocytes, e.g., T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells , eosinophils and basophils) in the tumor microenvironment can be reduced and/or attenuated by administering therapeutically effective amounts of an anti-αvβ8 integrin antibody of the invention to a subject (eg, a subject suffering from cancer).

In some cases, administration of an anti-αvβ8 integrin antibody or antigen-binding fragment thereof to a subject suffering from cancer increases the number (eg, immunohistochemical (IHC) density) of infiltrating immune cells, eg, total CD45 lymphocytes and myeloid cells, CD3 T-cells. cells, CD4 T cells, CD8 T cells and cells expressing GranzymeB (eg, activated CD8 and NK cells) in a tumor sample (eg, solid tumor sample) obtained from the subject. The increase in the number (eg, density) of infiltrating immune cells can be determined, for example, by immunohistochemistry (IHC) analysis of a tumor sample (eg, a solid tumor sample) obtained from a subject suffering from cancer compared with the number (eg, density) of infiltrating immune cells obtained from a reference tumor sample (e.g., a tumor sample obtained from the same subject or another subject suffering from a similar cancer, where the tumor sample was collected prior to administration (e.g., first administration or subsequent administration) of an anti-αvβ8 integrin antibody or antigen binding fragment thereof ).

In one aspect, the invention relates to treating a subject in vivo using an anti-αvβ8 integrin antibody or an antigen binding fragment thereof such that the growth of cancerous tumors is inhibited or reduced. In some embodiments, the subject treated in vivo with an anti-αvβ8 integrin antibody or antigen binding fragment thereof has a primary cancer, such as locally advanced cancer. In some embodiments, a subject treated in vivo with an anti-αvβ8 integrin antibody or antigen binding fragment thereof has a recurrent cancer, such as metastatic cancer.

In one aspect, the invention relates to in vivo treatment of a subject suffering from cancer or tumor that expresses αvβ8 integrin, β8 integrin (ITGβ8) and/or αV integrin (ITGαV), using an anti-αvβ8 integrin antibody or an antigen binding fragment thereof. In some embodiments, expression of αvβ8, β8 integrin and/or αV integrin is protein expression. In some embodiments, expression of αvβ8, β8 integrin and/or αV integrin is mRNA expression. In some embodiments, the expression of αvβ8, β8 integrin and/or αV integrin is increased expression relative to the level of expression of αvβ8, β8 integrin and/or αV integrin in the normal tissue or sample, in the control tissue or sample, in the tissue or sample before treatment and/ or tissue or sample after treatment. In one embodiment, the tissue or sample used to determine the relative expression levels of αvβ8, β8 integrin and/or αV integrin can be obtained from a subject with cancer or tumor, from another subject having the same type of cancer or tumor, or a different type of cancer or tumor or from a subject without cancer or tumor.

In some embodiments, αvβ8, β8 integrin, and/or αV integrin mRNA expression is increased expression relative to the level of expression in normal tissue or sample, control tissue or sample, pre-treatment tissue or sample, and/or post-treatment tissue or sample. In some embodiments, the mRNA expression of αvβ8, β8 integrin, and/or αV integrin is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% relative to the level of αvβ8, β8 integrin and/or αV integrin mRNA expression in a normal tissue or sample, in control tissue or sample, pre-treatment tissue or sample, and/or post-treatment tissue or sample.

An anti-αvβ8 integrin antibody or antigen-binding fragment thereof can be used alone (eg, as monotherapy) to inhibit cancer growth. Alternatively, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof may be used in combination with one or more of: a standard treatment (eg, for cancer or infectious disorders), another antibody or antigen-binding fragment thereof, an immunomodulator (eg, a costimulatory molecule activator or an inhibitory molecule inhibitor). molecules); vaccines, such as a therapeutic cancer vaccine; or other forms of cellular immunotherapy.

Therefore, in one embodiment, the invention provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an anti-αvβ8 integrin antibody or antigen binding fragment thereof described herein.

In one embodiment, the methods are suitable for treating cancer in vivo . To achieve enhanced immunity, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof can be administered together with an antigen of interest. When an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is administered in combination with one or more agents, the combination can be administered in any order or simultaneously.

In another aspect, a method is provided for treating a subject, for example , reducing or ameliorating a condition, hyperproliferative condition or disorder ( eg , cancer), such as a solid tumor, hematologic cancer, soft tissue tumor, or metastatic lesion, in the subject. The method involves administering to a subject one or more anti-αvβ8 integrin antibodies or antigen binding fragments thereof described herein, alone or in combination with other agents or therapeutic methods. In some embodiments, an anti-αvβ8 integrin antibody or antigen binding fragment thereof of the invention may be administered as 1st line therapy in the treatment of untreated subjects, or as 2nd line therapy following relapse or progression of cancer.

Exemplary cancers whose growth can be treated, eg, reduced, using the antibody molecules described herein include cancers typically sensitive to immunotherapy. Non-limiting examples of cancers for treatment include melanoma ( eg , metastatic malignant melanoma, cutaneous melanoma), renal cell carcinoma (RCC) ( eg , clear cell carcinoma, papillary cell carcinoma), prostate cancer ( eg , hormone-resistant prostate adenocarcinoma), breast cancer , ovarian cancer ( eg , epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer), head and neck cancer (eg, squamous cell carcinoma of the head and neck (SCCHN)), colon cancer, esophageal cancer ( eg , adenocarcinoma and squamous cell carcinoma) , stomach cancer ( eg , gastric and gastroesophageal junction adenocarcinoma), pancreatic cancer ( eg , pancreatic ductal adenocarcinoma), bile duct cancer ( eg , cholangiocarcinoma); endometrial cancer (eg, endometrial carcinoma), urothelial carcinoma, and lung cancer ( eg , non-small cell lung cancer, squamous cell carcinoma). In addition, refractory or relapsed malignancies can be treated with the antibody molecules described herein.

Examples of other cancers that may be treated include bone cancer, cutaneous or intraocular malignant melanoma, rectal cancer, anal cancer, testicular cancer, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Merkel cell cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic kidney syndromes or urethral cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmental cancers including asbestos-related cancers ( eg mesothelioma) and combinations of these cancers. In some embodiments, the cancer is selected from renal cell carcinoma, ovarian cancer, squamous cell carcinoma of the head and neck, and skin cancer (eg, melanoma, eg, advanced melanoma).

In some cases, the cancer is selected from the group consisting of a solid tumor, hematologic cancer (eg, leukemia, lymphoma, myeloma, eg multiple myeloma), and metastatic disease. Cancer can be a solid tumor, for example, a solid tumor such as malignant neoplasms, for example , sarcomas and carcinomas, for example , adenocarcinomas of various organ systems, such as lung ( for example , non-small cell lung cancer (NSCLC)), breast, ovarian, lymphoid, gastrointestinal intestinal tract ( eg colon), anal canal, genital and genitourinary tract ( eg kidney, urothelium, bladder cells, prostate), pharynx, central nervous system ( eg brain, nerve or glial cells), head and neck ( eg head and neck squamous cell carcinoma (HNSCC)), skin ( eg melanoma, eg advanced melanoma), pancreas, colon, rectum, kidney ( eg renal cell carcinoma), liver, small bowel cancer and esophageal cancer, esophageal junction cancer, thyroid cancer and cervical cancer. In some cases, the cancer may be a lymphoproliferative disorder ( eg , post-transplant lymphoproliferative disorder) or a hematologic cancer, T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or leukemia ( eg , myeloid leukemia or lymphoid leukemia). In some cases, the cancer is early-stage, intermediate-stage, late-stage, or metastatic cancer. In specific cases, the cancer is renal cell carcinoma, ovarian cancer, or squamous cell carcinoma of the head and neck.

In other embodiments, the cancer is a hematologic malignancy or cancer, including, but not limited to, leukemia or lymphoma. For example, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof can be used to treat cancers and malignancies, including, but not limited to, acute leukemias, including, but not limited to, e.g. , B-cell acute lymphoid leukemia (“BALL”) , T-cell acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL); one or more chronic leukemias, including, but not limited to, chronic myelogenous leukemia ( CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to, B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphomas ferrative conditions , MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström's macroglobulinemia and "preleukemia, which together represent a diverse collection of hematological conditions united by the ineffective production of ( or dysplasia) of myeloid blood cells and the like.

In one embodiment, the cancer is selected from lung cancer ( eg , non-small cell lung cancer (NSCLC) ( eg , NSCLC with squamous and/or non-squamous cell histology or NSCLC adenocarcinoma)), melanoma ( eg , advanced melanoma), skin squamous cell carcinoma (SCC) skin) ( eg , metastatic skin SCC), kidney cancer (eg, renal cell carcinoma, clear cell renal cell carcinoma), liver cancer, myeloma ( eg , multiple myeloma), prostate cancer, breast cancer ( eg , breast cancer, which does not express one, two, or all of the estrogen receptor, progesterone receptor, or Her2/neu, e.g. , triple-negative breast cancer), colorectal cancer, pancreatic cancer, head and neck cancer ( e.g. , head and neck squamous cell carcinoma (HNSCC), cancer anal canal, gastroesophageal junction cancer, thyroid cancer, cervical cancer, lymphoproliferative disease ( eg , post-transplant lymphoproliferative disease) or hematologic cancer, T-cell lymphoma, non-Hodgkin's lymphoma, or leukemia ( eg , myeloid leukemia).

In another embodiment, the cancer is selected from carcinoma ( eg , advanced or metastatic carcinoma), melanoma, or lung carcinoma, eg , non-small cell lung carcinoma.

In one embodiment, the cancer is lung cancer, for example non-small cell lung cancer.

In another embodiment, the cancer is prostate cancer, such as advanced prostate cancer.

In yet another embodiment, the cancer is myeloma, such as multiple myeloma.

In yet another embodiment, the cancer is a kidney cancer, such as renal cell carcinoma (RCC) ( eg , a metastatic RCC or clear cell renal cell carcinoma).

In some embodiments, the cancer is a skin cancer ( eg , melanoma, eg , advanced melanoma, eg, cutaneous squamous cell carcinoma, eg, metastatic cutaneous squamous cell carcinoma).

In one embodiment, the cancer is melanoma, such as advanced melanoma. In one embodiment, the cancer is advanced or inoperable melanoma that has not responded to other therapies.

In some embodiments, the cancer is kidney cancer, such as renal cell carcinoma (RCC). In some cases, the kidney cancer is metastatic RCC, clear cell renal cell carcinoma (ccRCC) or non-clear cell renal cell carcinoma (ncRCC). In some cases, if the cancer is RCC, for example, ccRCC, an anti-αvβ8 integrin antibody or antigen binding fragment thereof of the invention may be administered as 1st line or 2nd line therapy.

In some embodiments, if the cancer is ovarian cancer, an anti-αvβ8 integrin antibody or antigen binding fragment thereof of the invention may be administered as 2nd line therapy to platinum-resistant patients.

In some embodiments, the cancer is platinum-resistant and/or recurrent cancer.

The methods and compositions described herein are used to treat metastatic lesions associated with the above cancers.

Combination therapies

Tumors evade host immune surveillance through a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and are immunosuppressive. These include, among others, TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. & O'Garra, A. ( 1992) Immunology Today 13: 198-200) and Fas ligand (Hahne, M. et al. (1996) Science 274: 1363-1365). Antibodies or an antigen binding fragment thereof for each of these substances can be used in combination with an anti-αvβ8 integrin antibody or an antigen binding fragment thereof to counteract the effects of the immunosuppressive agent and promote antitumor immune responses in the host.

The antibodies and antigen binding fragments described herein can be used in unconjugated forms or conjugated to a second agent, eg, a cytotoxic drug, radioisotope, or protein, eg, a protein toxin or viral protein. This method includes: administering an antibody molecule, alone or conjugated to a cytotoxic drug, to a subject in need of such treatment. Antibody molecules can be used to deliver a variety of therapeutic agents, for example, a cytotoxic moiety, e.g., a therapeutic drug, a radioisotope, molecules of plant, fungal or bacterial origin, or biological proteins (e.g., protein toxins) or particles (e.g., recombinant viral particles, e.g.; through the viral envelope protein) or mixtures thereof.

In certain embodiments, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof may be used in combination with other therapies to produce an unexpected synergistic therapeutic effect that may provide an effect greater than simply the additive effect of adding two separate therapies. For example, a combination therapy may include a composition of the present invention formulated with and/or co-administered with one or more additional therapeutic agents, for example , one or more anticancer agents, cytotoxic or cytostatic agents, hormonal treatments, vaccines and/or other immunotherapies. In other embodiments, antibody molecules are administered in combination with other therapeutic treatments, including surgery, radiation, cryosurgery, and/or thermotherapy. Such synergistic combination therapies may advantageously utilize lower doses of therapeutic agents administered, thereby avoiding the potential toxicity or complications associated with different monotherapies.

In certain embodiments, the methods and compositions described herein are administered in combination with one or more other antibody molecules, chemotherapy, other anticancer therapies ( eg , targeted anticancer therapies or oncolytic drugs), cytotoxic agents, immune therapies ( eg , cytokines), surgical and/or radiation therapies. Typical cytotoxic agents that may be administered in combination include antimicrotubulin agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents capable of interacting with the signal transduction pathway, agents that promote apoptosis, proteasome inhibitors and radiation ( eg local or whole body irradiation).

Alternatively, or in combination with the above combinations, the method and composition described herein may be administered in combination with one or more of: an immunomodulator (e.g., a co-stimulatory molecule activator or an inhibitory molecule inhibitor); a vaccine, for example a therapeutic cancer vaccine; or other forms of cellular immunotherapy.

Exemplary non-limiting synergistic combinations and uses of anti-αvβ8 integrin antibodies or antigen binding fragments thereof include the following.

In certain embodiments, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered in combination with a costimulatory molecule modulator or inhibitory molecule, such as a co-inhibitory ligand or receptor.

In one embodiment, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is administered in combination with a modulator, such as a costimulatory molecule agonist. In one embodiment, the costimulatory molecule agonist is selected from an agonist ( eg , an agonist antibody or antigen binding fragment or soluble fusion protein thereof) 4-1BB (CD137), OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/ CD18), ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160 or B7-H3.

In another embodiment, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is used in combination with an immunomodulator, e.g., a co-stimulatory molecule, e.g. , an agonist or positive signal-associated modulator, which includes co-stimulatory domain 4-1BB (CD137), CD28, CD27 , ICOS and GITR.

Exemplary GITR modulators include, for example , GITR fusion proteins and anti-GITR antibodies ( e.g. , divalent anti-GITR antibodies), such as the GITR fusion proteins described in US Patent No.: 6,111,090, European Patent No.: 090505B1, US Patent No.: 8,586,023 , PCT publication nos: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, for example, in US Patent No: 7,025,962, European Patent No: 1947183B1, US Patent No: 7,812,135, US Patent No: 8,388,967, US Patent No: 8,591,886, European Patent No: EP 1866339, PCT Publication No: WO 2011/028683, PCT Publication No: WO 2013/039954, PCT Publication No: WO2005/007190, PCT Publication No: WO 2007/133822, PCT Publication ations No: WO2005/055808, PCT Publication No: WO 99/40196, PCT Publication No: WO 2001/03720, PCT Publication No: WO99/20758, PCT Publication No: WO2006/083289, PCT Publication No: WO 2005/115451, Patent US No: 7,618,632 and PCT Publication No: WO 2011/051726. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered in combination with an immune checkpoint molecule inhibitory molecule inhibitor. Those skilled in the art will understand that the term “immune checkpoints” refers to a group of molecules on the cell surface of CD4 and CD8 T cells. These molecules can effectively serve as “brakes” to suppress or inhibit the antitumor immune response. Immune checkpoint molecules include, but are not limited to, programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, LAG-3, and TIM -3, which directly inhibit immune cells. Immunotherapeutic agents that may act as immune checkpoint inhibitors useful in the methods of the present invention include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM, and/or TGF beta. Inhibition of an inhibitory molecule can be accomplished through inhibition at the DNA, RNA or protein level. In embodiments, inhibitory nucleic acids (eg, dsRNA, siRNA, or shRNA) can be used to inhibit expression of the inhibitory molecule. In other embodiments, the inhibitor of the inhibitory signal is a polypeptide, such as a soluble ligand or antibody, or an antigen-binding fragment thereof, that binds to the inhibitory molecule.

In one embodiment, the inhibitor is a soluble ligand ( eg , CTLA-4-Ig or TIM-3-Ig) or antibody or antibody fragment that binds PD-1, PD-L1, PD-L2, or CTLA4. For example, an anti-PD-1 antibody molecule may be administered in combination with an anti-CTLA-4 antibody, e.g. ipilimumab, for example , to treat cancer ( e.g. cancer selected from: melanoma, e.g. metastatic melanoma; lung cancer, e.g. non-small cell lung cancer; or prostate cancer). Exemplary anti-CTLA4 antibodies include tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206); and ipilimumab (CTLA-4 antibody, also known as MDX-010, CAS No. 477202-00-9). In one embodiment, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is administered after treatment, for example after treatment of melanoma, with an anti-CTLA4 antibody ( eg , ipilimumab) with or without a BRAF inhibitor ( eg , vemurafenib or dabrafenib). Typical doses of an anti-CTLA-4 antibody, such as ipilimumab, are approximately 3 mg/kg.

Other antibodies that can be used to activate a host immune response can be used in combination with an anti-αvβ8 integrin antibody or an antigen binding fragment thereof. They include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace the activity of T-cell helpers. (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in combination with an anti-αvβ8 integrin antibody or an antigen binding fragment thereof. Antibodies to T cell costimulatory molecules such as CTLA-4 (eg, US Patent No. 5,811,097), OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I et al (1997) Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff, A. et al (1999) Nature 397: 262-266) may also cause increased levels of T cell activation.

In certain embodiments, an anti-αvβ8 integrin antibody or antigen binding fragment thereof described herein is administered in combination with one or more PD-1, PD-L1 and/or PD-L2 inhibitors known in the art. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered concomitantly with an immune checkpoint inhibitor (e.g., an anti-PD-1, anti-PD-L1 and/or anti-PD-L2 antibody or antigen binding fragment thereof) to a subject , who had not previously received immune checkpoint inhibitor treatment. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered concomitantly with an immune checkpoint inhibitor (e.g., an anti-PD-1, anti-PD-L1 and/or anti-PD-L2 antibody or antigen binding fragment thereof) to a subject who has previously received treatment with an immune checkpoint inhibitor and whose cancer or tumor has progressed (eg, locally spread, metastasized). In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered biweekly (eg, every 2 weeks) for a 28-day course, and an immune checkpoint inhibitor is administered every 4 weeks on day 1 of each 28-day course. In one embodiment, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is administered biweekly for a 28-day course, and an immune checkpoint inhibitor (eg, anti-PD-1, anti-PD-L1, and/or anti-PD-1) L2 antibody or antigen-binding fragment thereof) is administered every 4 weeks on day 1 of each 28-day course to a subject who has cancer or a tumor. In one embodiment, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen binding fragment thereof described in PCT Publication No. WO2016/092419 (eg, RN888, referred to as PF-06801591 or sasanlimab). In one embodiment, the cancer or tumor is a squamous cell carcinoma, such as squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), breast cancer, or colon cancer.

The PD-1, PD-L1 and/or PD-L2 inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide. In some embodiments, the anti-PD-1 antibody is selected from MDX-1106, Merck 3475, or CT-011. In one embodiment, the anti-PD-1 antibody is nivolumab/Opdivo®, pembrolizumab/Keytruda®, spartalizumab, pidilizumab, tislelizumab, AMP-224, AMP-514, cemiplimab, or sasanlimab (RN888, mAb7, PF-06801591). In one embodiment, the anti-PD-L1 antibody is MEDI4736, MPDL3280A (YW243.55.s70), BMS-936559 (MDX-1105), atezolizumab/Tecentriq®, durvalumab/Imfizi®, or avelumab/Bavencio®. In one embodiment, the anti-PD-L1 antibody is not avelumab. In one embodiment, an anti-PD-1 antibody is described in PCT Publication No. WO2016/092419, including, but not limited to, mAb7 (also referred to as RN888, PF-06801591, or sasanlimab).

In some embodiments, the PD-1 inhibitor is an immunoadhesin ( e.g. , an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region ( e.g. , the Fc region of an immunoglobulin sequence). In some embodiments, The PD-1 inhibitor is AMP-224. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 binding antagonist is selected from YW243.55.s70 (also known as MPDL3280A , atezolizumab), MEDI-4736, MSB-0010718C or MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874. Antibody YW243.55.s70, also called MPDL3280A (heavy and light chain variable region sequences are shown in SEQ ID Nos. 20 and 21, respectively) is an anti-PD-L1 described in WO 2010/077634.

MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in WO2006/121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in WO2009/101611. In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (trade name KEYTRUDA, formerly labrolizumab, also known as MK-3475) is described, for example , in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44. AMP-224 (B7-DCIg; amplyimmune; e.g. described in WO2010/027827 and WO2011/066342) is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1. Other anti-PD-1 antibodies include AMP-514 (Amplyimmune), among others, for example the anti-PD-1 antibodies described in US 8,609,089, US 2010028330 and/or US 20120114649.

In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for MDX-1106 include MDX-1106-04, ONO-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4). Nivolumab (also called BMS-936558 or MDX1106; Bristol-Myers Squibb) is a full human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US 8,008,449 and WO2006/121168. Lambrolizumab (also called pembrolizumab or MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8,354,509 and WO2009/114335. MDPL3280A (Genentech/Roche) is an Fc optimized human IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A (also known as YW243,55.s70, and other human monoclonal antibodies to PD-L1 are described in U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906. Sequences of YW243,55.s70 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21) are also presented in WO2010/077634 and US 8,217.149). MDX-1105 (also called BMS-936559) and other anti-PD-L1 binding agents are described in WO2007/005874.

In other embodiments, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is administered in combination with a cytokine, such as interleukin-21, interleukin-2, interleukin-12, or interleukin-15. In certain embodiments, a combination of an anti-αvβ8 integrin antibody or an antigen binding fragment thereof and a cytokine described herein is used to treat a cancer, such as a cancer described herein ( eg , solid tumor or melanoma).

In all methods described herein, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof may be combined with other forms of immunotherapy, such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2, IL-21 ) or bispecific antibody therapy, which provides enhanced presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

Exemplary immunomodulators that may be used in combination with an anti-αvβ8 integrin antibody or antigen binding fragment thereof include, but are not limited to , afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimide (CC4047); and cytokines, eg IL-21 or IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon γ, CAS 951209-71-5, available from IRX Therapeutics).

In some embodiments, the cancer is ovarian cancer, and the anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered in combination with a PARP1 inhibitor (e.g., olaparib, rucaparib, niraparib, veliparib, iniparib, talazoparib, 3-aminobenzamide, CEP 9722, E7016, BSI -201, KU-0059436, AG014699, MK-4827 or BGB-290).

In some embodiments, the cancer is a head and neck cancer, such as head and neck squamous cell carcinoma. In some embodiments, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof is administered in combination with radiation therapy.

Diagnostic Application

Anti-αvβ8 integrin antibodies or an antigen binding fragment thereof of the present invention can also be used to detect and/or measure αvβ8 integrin or cells expressing αvβ8 integrin in a sample, for example, for diagnostic purposes. For example, an anti-αvβ8 integrin antibody or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of αvβ8 integrin. Exemplary diagnostic assays for αvβ8 integrin may include, for example, contacting a sample obtained from a patient with an anti-anti-αvβ8 integrin antibody or antigen binding fragment thereof of the invention, wherein the anti-αvβ8 integrin antibody or antigen binding fragment thereof is labeled with a detectable label or reporter molecule.

In one aspect, the present invention provides a diagnostic method for detecting the presence of the αvβ8 integrin protein in vitro ( eg , in a biological sample, such as a tissue biopsy, such as from cancer tissue) or in vivo (eg, in vivo imaging in a subject). The method includes: (i) contacting a sample with an antibody or an antigen-binding fragment thereof described herein, or administering to a subject an antibody or an antigen-binding fragment thereof; (optional) (ii) contact with a reference sample, e.g. a control sample ( e.g. a control biological sample such as plasma, tissue, biopsy or a control subject)); and (iii) detecting the formation of a complex between the antibody or an antigen binding fragment thereof and the sample or subject or control sample or subject, wherein a change, e.g. , a statistically significant change, in complex formation in the sample or subject relative to the control sample or subject indicates the presence of αvβ8 integrin in the sample. The antibody molecule may be directly or indirectly labeled with an analyte to facilitate detection of bound or unbound antibody. Suitable analytes include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials, as described above and described in more detail below.

The term "sample" with respect to samples used for detection of polypeptides includes, but is not limited to, cells, cell lysates, proteins or cell membrane extracts, biological fluids or tissue samples.

Formation of a complex between an antibody molecule and an αvβ8 integrin can be detected by measuring or imaging either a binding molecule bound to an αvβ8 integrin antigen or an unbound binding molecule. Conventional detection methods, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry, can be used. As an alternative to labeling the antibody or its antigen-binding fragment, the presence of αvβ8 integrin can be analyzed in a sample using a competitive immunoassay using standards labeled with the analyte and an unlabeled antibody molecule. In this assay, a biological sample, labeled standards, and an antibody or antigen-binding fragment thereof are combined and the amount of labeled standard bound to an unlabeled binding molecule is determined. The amount of αvβ8 integrin in a sample is inversely proportional to the amount of labeled standard bound to the antibody or antigen-binding fragment.

V. COMPOSITIONS

Also provided herein are pharmaceutical compositions containing an effective amount of the anti-αvβ8 integrin antibody described herein. Examples of such compositions, as well as methods for their composition, are also described herein. In some embodiments, the composition contains one or more anti-αvβ8 integrin antibodies. In some embodiments, the anti-αvβ8 integrin antibody recognizes αvβ8 integrin (eg, human, mouse, cynomolgus, or rat αvβ8 integrin). In some embodiments, the anti-αvβ8 integrin antibody is a humanized antibody. In some embodiments, the anti-αvβ8 integrin antibody is a chimeric antibody. In some embodiments, the anti-αvβ8 integrin antibody contains a constant region that is capable of triggering a desired immune response, such as antibody-mediated lysis or ADCC.

The compositions used herein may further contain pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil/water emulsion, and various types of wetting agents. The preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBSA) or normal (0.9%) saline. Compositions containing such carriers are formulated by conventional means (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

Acceptable carriers, excipients or stabilizers are non-toxic to recipients in dosages and concentrations and may contain buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

Anti-αvβ8 integrin antibodies and compositions thereof may also be used in combination with other agents, including an additional therapeutic agent (e.g., an immune checkpoint molecule inhibitor) that serves to enhance and/or complement the effectiveness of the agents.

The disclosure also provides compositions, including pharmaceutical compositions, containing polynucleotides encoding antibodies herein. In some embodiments, the composition contains an expression vector containing a polynucleotide encoding an antibody, as described herein. In some embodiments, the composition contains either or both of the polynucleotides of SEQ ID NO: 1 or 4. In some embodiments, the composition contains either or both of the polynucleotides of SEQ ID NO: 183 or 4. In some embodiments, the composition contains either or both polynucleotide of SEQ ID NO: 185 or 189. In some embodiments, the composition contains either or both of the polynucleotides of SEQ ID NO: 185 or 191.

In another aspect, the polynucleotide may encode VH, VL, and/or both VH and VL antibodies herein. That is, the composition contains one polynucleotide or more than one polynucleotide encoding an antibody or antigen-binding fragment thereof, as herein provided.

The pharmaceutical compounds herein may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids , hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The pharmaceutical composition herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions herein include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil and organic esters for injection, such as ethyl oleate. Proper flow can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be achieved both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the pharmaceutical injection form can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutical compositions generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it will be suitable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols such as mannitol, sorbitol or sodium chloride. Prolonged absorption of the injection composition can be achieved by including in the composition an absorption delaying agent, for example, monostearate salts and gelatin.

Sterile injection solutions can be prepared by adding the active compound in the required amount to an appropriate diluent with one or a combination of the ingredients listed above, if required, followed by sterilization microfiltration.

Typically, dispersions are prepared by adding the active compound to a sterile vehicle that contains the basic dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredient from a pre-sterilized solution by filtration.

The pharmaceutical composition of the present disclosure may be formulated, packaged or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops, including, for example, a 0.1%-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further contain buffering agents, salts, or one or more other additional ingredients described herein. Other ophthalmologically administered formulations that may be used include those that contain the active ingredient in microcrystalline form or in a liposomal formulation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricants; sweeteners; flavorings; dyes; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oil carriers and solvents; suspending agents; dispersing or wetting agents; emulsifiers, softeners; buffers; salt; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions herein are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, PA (1985), which is included incorporated into this document by reference.

In one embodiment, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is formulated in a vial containing 100 mg of the anti-αvβ8 integrin antibody or antigen binding fragment thereof in 1 ml of an aqueous buffered solution.

In one embodiment, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered in an intravenous formulation as a sterile aqueous solution containing about 0.1 mg/ml, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml , about 4 mg/ml, 5 mg/ml, or in some embodiments, about 10 mg/ml, or in some embodiments, about 15 mg/ml, or in some embodiments, about 20 mg/ml antibody, or in some embodiments, about 25 mg/ml, or in some embodiments, about 50 mg/ml, or in some embodiments, about 100 mg/ml and 5% dextrose. In some embodiments, the intravenous formulation is a sterile aqueous solution containing from 0.1 mg/ml to 15 mg/ml anti-αvβ8 integrin antibody or antigen binding fragment thereof in 5% dextrose. In some embodiments, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is formulated in a composition containing sodium acetate, polysorbate 80 and sodium chloride at a pH of about 5 to 6. In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg/ml antibody, with 20 mM sodium acetate, 0.2 mg/ml polysorbate 80 and 140 mM sodium chloride at pH 5.5. Additionally, the solution containing the antibody or antigen binding fragment thereof may contain, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine and any combination thereof and many other compounds known in the art. areas.

In one embodiment, the pharmaceutical composition of the present disclosure contains the following components: 50 mg/ml anti-αvβ8 integrin antibody or antigen binding fragment thereof as described herein, 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA at pH 5.8; in another embodiment, the pharmaceutical composition of the present invention includes the following components: 100 mg/ml anti-αvβ8 integrin antibody or antigen binding fragment thereof as described herein, 10 mM histidine, 5% sucrose and 0.01% polysorbate 80 at pH 5.8 . This composition may be presented as a liquid formulation or as a lyophilized powder. When restored to full volume, the composition retains the same composition. Alternatively, the powder can be reconstituted to half volume, in which case the composition contains 100 mg of an anti-αvβ8 integrin antibody or antigen binding fragment thereof as described herein, 20 mM histidine, 10% sucrose and 0.02% polysorbate 80 at pH 5.8.

In one embodiment, a portion of the dose is administered by intravenous bolus and the remainder is administered by infusion of the antibody composition. For example, a 0.01 mg/kg intravenous injection of an anti-αvβ8 integrin antibody or antigen binding fragment thereof may be administered as a bolus, and the remaining dose of antibody may be administered by intravenous injection. A predetermined dose of anti-αvβ8 integrin antibody or antigen binding fragment thereof can be administered, for example, from one and a half to two hours to five hours.

With respect to the therapeutic agent, if the agent is, for example, a small molecule, it may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, for example, in combination with a physiologically acceptable cation or anion, as is well known in the art.

The pharmaceutical compositions described herein can be prepared by any method known or hereinafter developed in the field of pharmacology. In general, such production methods include the step of combining the active ingredient with a carrier or one or more other optional ingredients and then, if necessary or desired, molding or packaging the product into the desired single or multiple dosage form.

In one embodiment, the compositions herein are pyrogen-free formulations that are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are found within a microorganism and are released when the microorganism breaks down or dies. Pyrogens also include fever-inducing heat-stable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension, and shock when administered to humans. Because of the potential for harmful effects, it is beneficial to remove even small amounts of endotoxins from IV pharmaceutical drug solutions. The Food and Drug Administration (“FDA”) has established an upper limit of 5 endotoxin units (EU) per dose per kilogram of body weight per hour for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 ( 2000)). When therapeutic proteins are administered in quantities of several hundred or thousands of milligrams per kilogram of body weight, it is beneficial to remove even trace amounts of endotoxin. In one embodiment, the levels of endotoxin and pyrogen in the composition are less than 10 EU/mg or less than 5 EU/mg or less than 1 EU/mg or less than 0.1 EU/mg or less than 0.01 EU/mg or less than 0.001 EU/mg. In another embodiment, the levels of endotoxin and pyrogen in the composition are less than about 10 EU/mg or less than about 5 EU/mg or less than about 1 EU/mg or less than about 0.1 EU/mg or less than about 0 .01 EU/mg or less than about 0.001 EU/mg.

In one embodiment, the description includes administration of the composition, wherein said administration is oral, parenteral, intramuscular, intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal, intravenous, dermal, subcutaneous, or transdermal.

In another embodiment, the description further includes administering the composition in combination with other therapies, such as surgery, chemotherapy, hormonal therapy, biological therapy, immunotherapy, or radiation therapy.

VI. DOSING / ADMINISTRATION

To prepare pharmaceutical or sterile compositions comprising an anti-αvβ8 integrin antibody or an antigen binding fragment thereof herein, the antibody is mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents can be prepared by admixture with physiologically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, suspensions, aqueous solutions, lotions or suspensions (see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993 ) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

The choice of regimen for administering a therapeutic agent depends on several factors, including the metabolism of the substance in the subject's serum or tissue, the extent of symptoms, the immunogenicity of the substance, and the availability of target cells in the biological matrix. In certain embodiments, the administration schedule maximizes the amount of therapeutic drug delivered to the patient, consistent with an acceptable level of side effects. Therefore, the amount of biological agent delivered depends in part on the specific agent and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies, cytokines and small molecules are available (see, for example, Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.).1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New Engl. J. Med. 348: 601-608; Milgrom, et al., 1999, New Engl. J. Med. 341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792; Beniaminovitz, et al. ., 2000, New Engl. J. Med. 342:613-619; Ghosh, et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000, New Engl. J. Med. Med 343:1594-1602).

Determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art to affect or be predicted to affect treatment. Typically, the dose starts at an amount slightly less than the optimal dose and is then increased in small increments until the desired or optimal effect relative to any negative side effects is achieved. Important diagnostic measures include symptoms, such as inflammation or levels of inflammatory cytokines produced.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration without being toxic to the patient. The dosage level selected will depend on a number of pharmacokinetic factors, including the potency of the specific compositions used herein or their ester, salt or amide, route of administration, time of administration, elimination rate of the particular compound used, duration of treatment, other drugs, compounds and/or materials. , used in combination with the particular compositions employed, the age, sex, body weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

Compositions containing anti-αvβ8 integrin antibodies or an antigen binding fragment thereof herein can be delivered by continuous infusion or by dosing at intervals, for example, one day, one week, or 1-7 times per week. Doses may be administered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. A specific dosing protocol is one involving the maximum dose or dosing frequency that avoids significant unwanted side effects. The total weekly dose may be at least 0.05 mcg/kg body weight at least 0.2 mcg/kg at least 0.5 mcg/kg at least 1 mcg/kg at least 10 mcg/kg at least at least 100 µg/kg at least 0.2 mg/kg at least 1.0 mg/kg at least 2.0 mg/kg at least 10 mg/kg at least 15 mg/kg at least 20 mg/kg at least 25 mg/kg or at least 50 mg/kg (see, e.g., Yang, et al., 2003, New Engl. J. Med. 349:427-434; Herold, et al. ., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003, Cancer. Immunol. Immunother 52: 133-144). The dose may be at least 15 mcg at least 20 mcg at least 25 mcg at least 30 mcg at least 35 mcg at least 40 mcg at least 45 mcg at least 50 mcg at least 55 mcg at least at least 60 µg at least 65 µg at least 70 µg at least 75 µg at least 80 µg at least 85 µg at least 90 µg at least 95 µg or at least 100 µg. The number of doses administered to a subject may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more.

For the anti-αvβ8 integrin antibodies or antigen binding fragment thereof herein, the dose administered to the patient may be from 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dose may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0 .0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, from 0.0001 mg/kg to 0.25 mg/kg , from 0.0001 to 0.15 mg/kg, from 0.0001 to 0.10 mg/kg, from 0.001 to 0.5 mg/kg, from 0.01 to 0.25 mg/kg or from 0, 01 to 0.10 mg/kg patient body weight. In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient is about 0.1 mg/kg, about 0.3 mg/kg, about 2 mg/kg, or about 3.0 mg/kg body weight patient's body. In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof is about 0.4 mg/kg, about 4 mg/kg, about 40 mg/kg, or about 100 mg/kg body weight patient's body.

In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof every 14 days is from about 1 mg/kg to about 12 mg/kg of the patient's body weight. In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof every 14 days is approximately 2 mg/kg of the patient's body weight. In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof every 14 days is approximately 7 mg/kg of the patient's body weight.

In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof every 28 days is from about 1 mg/kg to about 20 mg/kg of the patient's body weight. In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof every 28 days is approximately 4 mg/kg of the patient's body weight. In some embodiments, the dose of an anti-αvβ8 integrin antibody or antigen binding fragment thereof administered to a patient in need thereof every 28 days is approximately 12 mg/kg of the patient's body weight.

The dose of an anti-αvβ8 integrin antibody or antigen-binding fragment thereof can be calculated using the patient's body weight in kilograms (kg) multiplied by the administered dose in mg/kg. The dose of the antibody herein may be 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 µg/kg or less, 75 µg/kg or less, 70 µg/kg or less, 65 µg/kg or less, 60 µg/kg or less, 55 µg/kg or less, 50 µg/kg or less, 45 µg /kg or less, 40 mcg/kg or less, 35 mcg/kg or less, 30 mcg/kg or less, 25 mcg/kg or less, 20 mcg/kg or less, 15 mcg/kg or less, 10 mcg/ kg or less, 5 mcg/kg or less, 2.5 mcg/kg or less, 2 mcg/kg or less, 1.5 mcg/kg or less, 1 mcg/kg or less, 0.5 mcg/kg or less than or 0.1 mcg/kg or less than the patient's body weight.

The unit dose of anti-αvβ8 integrin antibodies or antigen binding fragment thereof herein may be from 0.1 mg to 200 mg, from 0.1 mg to 175 mg, from 0.1 mg to 150 mg, from 0.1 mg to 125 mg, from 0.1 mg to 100 mg, from 0.1 mg to 75 mg, from 0.1 mg to 50 mg, from 0.1 mg to 30 mg, from 0.1 mg to 20 mg, from 0, 1 mg to 15 mg, from 0.1 mg to 12 mg, from 0.1 mg to 10 mg, from 0.1 mg to 8 mg, from 0.1 mg to 7 mg, from 0.1 mg to 5 mg , from 0.1 to 2.5 mg, from 0.25 mg to 20 mg, from 0.25 to 15 mg, from 0.25 to 12 mg, from 0.25 to 10 mg, from 0.25 to 8 mg, from 0.25 mg to 7 mg, from 0.25 mg to 5 mg, from 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg or 1 mg to 2.5 mg. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered at a unit dose of about 100 mg, about 300 mg, about 500 mg, about 600 mg, about 800 mg, about 1200 mg, about 1400 mg, or about 1600 mg.

The dosage of the anti-αvβ8 integrin antibody or antigen binding fragment thereof herein may achieve a serum titer of at least 0.1 μg/ml at least 0.5 μg/ml at least 1 μg/ml at least 2 μg/ml by at least 5 µg/ml at least 6 µg/ml at least 10 µg/ml at least 15 µg/ml at least 20 µg/ml at least 25 µg/ml at least 50 µg/ml at least at least 100 µg/ml at least 125 µg/ml at least 150 µg/ml at least 175 µg/ml at least 200 µg/ml at least 225 µg/ml at least 250 µg/ml at least 275 µg/ml at least 300 µg/ml at least 325 µg/ml at least 350 µg/ml at least 375 µg/ml or at least 400 µg/ml in the subject. Alternatively, the dosage of antibodies herein may achieve a serum titer of at least 0.1 μg/ml at least 0.5 μg/ml at least 1 μg/ml at least 2 μg/ml at least 5 μg/ml at least 6 µg/ml at least 10 µg/ml at least 15 µg/ml at least 20 µg/ml at least 25 µg/ml at least 50 µg/ml at least 100 µg/ml at least 125 µg/ml at least 150 µg/ml at least 175 µg/ml at least 200 µg/ml at least 225 µg/ml at least 250 µg/ml at least 275 µg/ml at least at least 300 µg/ml at least 325 µg/ml at least 350 µg/ml at least 375 µg/ml or at least 400 µg/ml in the subject.

Doses of the anti-αvβ8 integrin antibody or antigen binding fragment thereof herein may be repeated, and administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months , 75 days, 3 months or at least 6 months.

The effective amount for a particular patient may vary depending on factors such as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FIa.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

The route of administration may be, for example, topical or dermal application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or sustained release or implant systems (see, for example, Sidman et al., 1983 , Biopolymers 22:547-556; Langer, et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem. Tech. 12:98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; US Patent Nos. 6,350466 and 6,316,024) . In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered intravenously. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered subcutaneously.

If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. In addition, pulmonary administration, for example, using an inhaler or nebulizer and an aerosol agent formulation, can also be used. See, for example, US Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, an anti-αvβ8 antibody or antigen binding fragment or composition thereof is administered using Alkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

The composition of the present disclosure may also be administered by one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by one skilled in the art, the route and/or method of administration will vary depending on the desired results. Selected routes of administration of the antibody herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. Parenteral administration may be methods of administration other than enteral and local administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular , subarachnoid, intraspinal, epidural and intrasternal, injection and infusion. Alternatively, the composition herein may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered as a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed Eng. 14:20; Buchwald et al. 1980, Surgery 88:501; Saudek et al. 1989, N. Engl. J. Med. 321:514).

Polymeric materials can be used to achieve controlled or delayed release of therapeutic agents herein (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FL. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. ScL Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 11 225:190; During et al., 19Z9, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71: 105); US Patent No. 5,679,377; US Patent No. 5,916,597; US Patent No. 5,912,015; US Patent No. 5,989,463; US Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), polyvinyl alcohol), polyacrylamide, polyethylene glycol), polylactides (PLA), polyoactide-co-glycolide copolymers (PLGA) and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, shelf stable, sterile, and biodegradable. A controlled or sustained release system can be placed adjacent to a prophylactic or therapeutic target, requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984 )).

Controlled release systems are discussed in review in Langer, 1990, Science 249:1527-1533. Any technique known to one skilled in the art can be used to prepare sustained release formulations containing one or more antibodies herein or conjugates thereof. See, for example, US Patent No. 4,526,938, International Patent Publications Nos. WO 91/05548, WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy and Oncology 59:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science and Technology 50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Ml. Symp. Control. Rel. Bioact. Mater. 24:853-854 and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Ml. Symp. Control Rel. Bioact. Mater. 24:759-160, each of which is incorporated herein by reference in its entirety.

If the anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered topically, it may be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion or other form well known to one skilled in the art. in this area. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). Non-sprayable topical dosage forms typically employ viscous or semi-solid or solid forms containing a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some cases greater than that of water. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, balms and the like, which, if desired, are sterilized or mixed with auxiliary agents (for example, preservatives, stabilizers, wetting agents, buffers or salts) to influence various properties, such as, for example, osmotic pressure. Other suitable dosage forms for topical use include nebulized aerosol preparations, where the active ingredient, in some cases in combination with a solid or liquid inert carrier, is packaged in a mixture with a volatile substance under pressure (for example, a gaseous propellant such as freon) or in a soft bottle. If desired, humectants or wetting agents may also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

When compositions containing anti-αvβ8 integrin antibodies or an antigen-binding fragment thereof are administered intranasally, they may be formulated as an aerosol, spray, fine mixture, or droplet form. In particular, prophylactic or therapeutic agents for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray from pressure packs or a nebulizer using a suitable propellant gas (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas) . In the case of a pressurized aerosol, the dosage form may be determined by a valve to deliver a metered amount. Capsules and cartridges (consisting, for example, of gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

Methods of co-administration or treatment with an additional therapeutic agent, such as an immune checkpoint molecule, a cytokine, a steroid, a chemotherapeutic agent, an antibiotic, or radiation therapy, are well known in the art (see, e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams and Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams and Wilkins, Phila., Pa.).

An effective amount of the therapeutic agent can reduce symptoms by at least 10 percent; by at least 20 percent; at least about 30 percent; at least 40 percent or at least 50 percent.

Additional therapies (eg, prophylactic or therapeutic agents) that can be administered in combination with anti-αvβ8 integrin antibodies or antigen binding fragments herein can be administered at an interval of less than 5 minutes, at an interval of less than 30 minutes, at an interval of 1 hour, with at intervals of about 1 hour, at intervals from about 1 to about 2 hours, at intervals from about 2 hours to about 3 hours, at intervals from about 3 hours to about 4 hours, at intervals from about 4 hours to about 5 hours, at intervals from about 5 hours to about 6 hours, from about 6 hours to about 7 hours, from about 7 hours to about 8 hours, from about 8 hours to about 9 hours, from about 9 hours to about 10 hours, at intervals from about 10 hours to about 11 hours, at intervals from about 11 hours to about 12 hours, at intervals from about 12 hours to 18 hours, at intervals from 18 hours to 24 hours, at intervals from 24 hours to 36 hours, at intervals from 36 hours to 48 hours, at intervals from 48 hours to 52 hours, at intervals from 52 hours to 60 hours, at intervals from 60 hours to 72 hours, at intervals from 72 hours to 84 hours, at intervals from 84 hours to 96 hours or at intervals from 96 hours to 120 hours from the antibodies herein. Two or more therapies may be administered during a single patient visit.

Methods for administering antibody molecules are known in the art and are described below. Suitable dosages of the molecules used will depend on the age and body weight of the subject and the particular drug used. Dosages and treatment regimens for anti-αvβ8 integrin antibody or its antigen-binding fragment, antibody molecule can be determined by a qualified specialist. In certain embodiments, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 10 to about 14 mg/kg, about 5 to 9 mg/kg, about 7 mg/kg, or about 12 mg/kg. In some embodiments, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered at a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg /kg, approximately 7 mg/kg, approximately 8 mg/kg, approximately 9 mg/kg or 10 mg/kg, approximately 11 mg/kg, approximately 12 mg/kg, approximately 13 mg/kg, approximately 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 40 mg/kg. In some embodiments, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered at a dose of about 1-5 mg/kg, about 5-10 mg/kg, or about 10-15 mg/kg. In some embodiments, the anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered at a dose of about 0.5-2, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2- 10, 2-15, 5-15 or 5-20 mg/kg.

The dosage schedule may vary from, for example, once a week to once every 2, 3, 4, 5 or 6 weeks. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered at a dose of about 10 to 20 mg/kg (e.g., about 7 mg/kg or about 12 mg/kg) every other week (e.g., every two weeks or once in two weeks). In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof is administered at a dose of about 10 to 20 mg/kg (e.g., about 7 mg/kg or about 12 mg/kg) once a month (e.g., every four weeks ). In some embodiments, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing it, is administered intravenously. In some embodiments, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, or a pharmaceutical composition containing it, is administered subcutaneously.

In some embodiments, an anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered intravenously or subcutaneously every two weeks. In one embodiment, an anti-αvβ8 integrin antibody or antigen binding fragment thereof herein is administered at a unit dose of about 100 mg, about 300 mg, about 800 mg, about 1200 mg, or about 1600 mg intravenously or subcutaneously every two weeks. In some embodiments, the subject is administered an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof herein at a unit dose of about 100 mg, about 300 mg, about 800 mg, about 1200 mg, or about 1600 mg intravenously every two weeks and is administered anti- PD1 inhibitor every four weeks. In some embodiments, the anti-PD1 inhibitor antibody is administered subcutaneously at a standard dose of 300 mg. In some embodiments, the anti-PD1 inhibitor is an anti-PD1 antibody described in PCT Publication No. WO2016/092419 (eg, mAb7, also referred to as RN888, PF-06801591, or sasanlimab).

In some embodiments, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing it, is administered about twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months , once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, one once every eleven months or once every twelve months. In some embodiments, an anti-αvβ8 integrin antibody or antigen binding fragment thereof, or a pharmaceutical composition containing it, is administered every two weeks, for example, up to 12 times (for example, up to 10, 8, 6, 5, 4, or 3 times). In some embodiments, an anti-αvβ8 integrin antibody or antigen binding fragment thereof, or a pharmaceutical composition containing it, is administered every four weeks, for example, up to 12 times (for example, up to 10, 8, 6, 5, 4, or 3 times).

In some embodiments, each administration of an anti-αvβ8 integrin antibody or antigen-binding fragment thereof contains 5-10 mg/kg (e.g., 5, 6, 7, 8, 9, or 10 mg/kg) of the antibody or antigen-binding fragment thereof, e.g., each administration contains approximately 7 mg/kg.

In some embodiments, the antibody or antigen binding fragment thereof is administered every four weeks, for example, up to 6 times (eg, up to 6, 5, 4, 3, 2 or 1 time).

In other embodiments, each administration of an anti-αvβ8 integrin antibody or antigen-binding fragment thereof contains 10-15 mg/kg (e.g., 10, 11, 12, 13, 14, or 15 mg/kg) of the antibody or antigen-binding fragment thereof, e.g., each administration contains approximately 12 mg/kg.

Anti-αvβ8 antibodies or an antigen binding fragment thereof herein and other therapeutic agents can be administered cyclically. Cyclic therapy includes administration of a first therapy (eg, a first prophylactic or therapeutic agent) for a specified period of time, followed by administration of a second therapy (eg, a second prophylactic or therapeutic agent) for a specified period of time, optionally followed by administration of a third therapy (eg , prophylactic or therapeutic agent) for a certain period of time and so on, and repeating this sequential administration, i.e. cycle to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and/or to improve the effectiveness of the therapies.

In certain embodiments, an anti-αvβ8 antibody or an antigen binding fragment thereof herein may be formulated to ensure proper distribution in vivo . For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds herein penetrate the BBB (if desired), they can be formulated, for example, in liposomes. Methods for making liposomes are presented, for example, in US Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more groups that are selectively transported to certain cells or organs, thereby improving targeted drug delivery (see, for example, VV Ranade, 1989, J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, for example, US Pat. No. 5,416,016); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PG Bloeman et al., 1995, FEBS Lett. 357: 140; M. Owais et al., 1995, Antimicrob. Agents Chemother. 39: 180); Surfactant protein receptor A (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen, 1994, FEBS Lett. 346:123; Killion; Fidler, 1994; Immunomethods 4:273.

The description provides protocols for administering a pharmaceutical composition containing anti-αvβ8 antibodies or an antigen binding fragment thereof herein, alone or in combination with other therapies, to a subject in need thereof. The therapies (eg, prophylactic or therapeutic agents) of the combination therapies of the present disclosure may be administered to a subject simultaneously or sequentially. Therapies (eg, prophylactic or therapeutic agents) of the combination therapies of the present disclosure may also be administered systemically.

The therapeutic agents (eg, prophylactic or therapeutic agents) of the combination therapy herein can be administered to a subject simultaneously. The term “concurrently” is not limited to administering therapeutic agents (eg, prophylactic or therapeutic agents) at the same time, but rather means that a pharmaceutical composition comprising an anti-αvβ8 integrin antibody or an antigen binding fragment thereof herein is administered to a subject sequentially and within such a time interval that the antibodies herein or their conjugates can act in conjunction with other therapy(s) to obtain increased benefit than if they were otherwise administered. For example, each therapy may be administered to a subject simultaneously or sequentially in any order at different time points; however, if they are not administered simultaneously, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any suitable form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to the subject at an interval of less than 15 minutes, less than 30 minutes, an interval of less than 1 hour, an interval of about 1 hour, an interval of about 1 hour to about 2 hours , at intervals from about 2 hours to about 3 hours, at intervals from about 3 hours to about 4 hours, at intervals from about 4 hours to about 5 hours, at intervals from about 5 hours to about 6 hours, at intervals from about 6 hours to about 7 hours, at intervals from about 7 hours to about 8 hours, at intervals from about 8 hours to about 9 hours, at intervals from about 9 hours to about 10 hours, at intervals from about 10 hours to about 11 hours, at an interval of about 11 hours to about 12 hours, at an interval of 24 hours, at an interval of 48 hours, at an interval of 72 hours, or at an interval of 1 week. In other embodiments, two or more therapies (eg, prophylactic or therapeutic agents) are administered during a single patient visit.

The prophylactic or therapeutic agents of the combination therapies may be administered to a subject in a single pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies may be administered to a subject simultaneously in separate pharmaceutical compositions. Prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

VII. SETS

Also provided herein are kits containing any or all of the antibodies described herein. The kits herein include one or more containers containing an anti-αvβ8 integrin antibody or antigen binding fragment thereof described herein and instructions for use in accordance with any of the methods herein described herein. Typically, these instructions describe the administration of an antibody for the therapeutic treatment described above. In some embodiments, kits are provided for preparing a single dose. In certain embodiments, the kit may contain both a first container containing a dried protein and a second container containing an aqueous formulation. In certain embodiments, kits containing an applicator are included, such as single-chamber and multi-chamber prefilled syringes (eg, liquid syringes and lyophilisate syringes).

Instructions related to the use of an anti-αvβ8 integrin antibody or antigen-binding fragment thereof generally include information regarding the dosage, dosage regimen, and route of administration for the intended treatment. Containers may be unit doses, bulk units (eg, multi-dose units), or subunit units. The instructions included in kits hereunder are typically written instructions on a label or package insert (e.g., a piece of paper included with the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also applicable.

The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Also provided are packages for use in combination with a specific device, such as an inhaler, a nasal administration device (eg, a nebulizer), or an infusion device such as a minipump. The kit may have a sterile access port (eg, the container may be an intravenous solution bag or a vial having a stopper that can be pierced with a hypodermic needle). At least one active agent in the composition is an anti-αvβ8 integrin antibody as provided herein. The container may further contain an additional therapeutic agent, as described herein.

The kit may further contain at least one anti-PD1 antibody, such as, but not limited to, nivolumab, pembrolizumab, spartlizumab, pidilizumab, tislelizumab, cemiplimab, sasanlimab (mAb7, RN888, PD-06801591), AMP-224, AMP- 514.

Sets may optionally include additional components, such as buffers, and explanatory information. Typically the kit includes a container and a label or insert(s) on or associated with the container.

The disclosure also provides diagnostic kits containing any or all of the antibodies or antigen binding fragments thereof described herein. Diagnostic kits are useful, for example, to detect the presence of αvβ8 integrin in a sample. In some embodiments, a diagnostic kit may be used to identify an individual with an underlying disease, disorder, or condition that may place the individual at risk for developing an αvβ8 integrin-mediated disease, disorder, or condition, or a disease, disorder, or condition associated with αvβ8 integrin deficiency. In some embodiments, the diagnostic kit can be used to detect the presence and/or level of αvβ8 integrin in an individual who is suspected of having an αvβ8 integrin-mediated disease, or a disease, disorder or condition associated with αvβ8 integrin deficiency.

Diagnostic kits herein include one or more containers containing an anti-αvβ8 integrin antibody or antigen binding fragment thereof described herein and instructions for use in accordance with any of the methods herein described herein. Typically, these instructions describe the use of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof to detect the presence of αvβ8 integrin in individuals at risk for or suspected of having an αvβ8 integrin-mediated disease or a disease, disorder or condition caused by αvβ8 integrin deficiency. In some embodiments, a typical diagnostic kit may be configured to contain reagents, such as, for example, an anti-αvβ8 integrin antibody or antigen binding fragment thereof, a negative control sample, a positive control sample, and instructions for use of the kit.

VIII. EQUIVALENTS

The above description and the following examples describe in detail certain specific embodiments herein and describe the best mode contemplated by the inventors. However, it should be appreciated that no matter how detailed the foregoing may be set forth in the text, the disclosure may be put into practice in many ways, and the disclosure should be construed in accordance with the appended claims and any equivalents thereof.

Although the disclosed teachings have been described with reference to various uses, methods, kits and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein and the claims below. The following examples are presented to better illustrate the ideas disclosed and are not intended to limit the scope of the ideas presented herein. Although the present teachings have been described in terms of these exemplary embodiments, one skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present invention.

All references cited herein, including patents, patent applications, articles, textbooks and the like, and references cited herein, to the extent not already included, are incorporated herein by reference in their entirety. To the extent that one or more of the included literature and similar materials differ from or are inconsistent with this application, including, but not limited to, certain terms, usage of terms, methods described and the like, they are controlled by this application.

IX. GENERAL METHODS

It should be understood that the present invention is not limited to the specific synthesis methods for production, which, of course, may vary. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. In addition, unless the context otherwise requires, terms in the singular shall include the plural and terms in the plural shall include the singular. Generally, the nomenclatures used in connection with the methods, cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry and protein and nucleic acid hybridization described herein are well known and widely used in the art.

The practice of the present invention will use, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the competence of those skilled in the art. Such methods are fully described in the literature, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Enzymatic reactions and purification methods are performed in accordance with the manufacturer's specifications, as is routinely done in the art or as described herein. The nomenclatures used in connection with the laboratory procedures and methods of analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein are well known and widely used in the art. Standard methods are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

X. BIOLOGICAL DEPOSITION

Type materials of the present invention are deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA February 13, 2018. Vector ADWA11 VH05-02-VH, having ATCC accession number PTA-124917, contains a DNA insert encoding the heavy chain variable region of the antibody ADWA11 2.4, also known as VH05-2_VK01(2.4) and ADWA11 5 -2 2.4. The ADWA11 VK2.4-VL vector, having ATCC accession number PTA-124918, contains a DNA insert encoding the light chain variable region of the ADWA11 2.4 antibody, also known as VH05-2_VK01(2.4) and ADWA11 5-2 2.4.

The deposits were made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This ensures that the deposit culture remains viable for a period of 30 years from the date of deposit. The deposit will be available at ATCC under the terms of the Budapest Treaty and subject to the agreement between Pfizer Inc. and ATCC, which guarantees the continued and unrestricted availability of the progeny of a culture deposit to the public upon the issuance of a corresponding U.S. patent or upon publication of any U.S. or foreign patent application, whichever occurs first, and guarantees the availability of the progeny to whoever is designated by the U.S. Commissioner of Patents and Trademarks as authorized to do so by 35 U.S.C. Section 122 and related Commissioner Rules (including 37 C.F.R. Section 1.14 with specific reference to 886 OG 638).

The owner of this application has agreed that if a culture of the deposited materials dies, is lost or is destroyed during cultivation under suitable conditions, the materials will be promptly replaced, upon notification, by others of the same kind. The availability of materials deposited shall not be construed as a license to use the invention in violation of rights granted under the authority of any government under its patent laws.

EXAMPLES

The disclosure is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise noted. Accordingly, the disclosure should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations that become apparent as a result of the ideas presented herein.

Example 1: Generation of anti- α v β 8 integrin antibodies to mouse hybridoma

Mouse hybridoma antibodies against human αvβ8 integrin are generated according to methods generally described in US Patent No. 9,969,804, which is incorporated herein by reference in its entirety.

Briefly, β8 integrin knockout mice that have been bred to unrelated CD1 individuals to ensure postnatal survival are immunized with recombinant human αvβ8 integrin (R&D Systems, 4135-AV-050) at a dose of 50 μg per mouse every two weeks until acceptable anti-integrin titers are obtained. αvβ8 antibodies. Serum from immunized mice is then screened by enzyme-linked assay to identify mice for hybridoma generation.

Antibodies from the generated hybridomas were then characterized by flow cytometry using SW480 cells transfected to express integrin αvβ8 or αvβ3 or αvβ6 as negative controls. Sw480 cells generally do not express any αv integrins, with the exception of αvβ5. The mouse hybridoma antibody ADWA-11 (also called ADWA11) was identified, and the specificity of this antibody was confirmed by flow cytometry in each cell line using labeled ADWA-11 or an antibody to αvβ5 (Alula) or αvβ3 (Axum-2) or αvβ6 (10D5) ( Su et al., Am J Respir Cell Mol Biol 36:377-386, 2007 Su et al Am J Respir Cell Mol Biol 185: 58-66, 2012 Huang et al. al., J Cell Sci 111 (Pt 15): 2189-2195).

Cell adhesion assays are also performed with U251 cells that express αvβ8 integrin on plates coated with 1 μg/ml TGFβ1 latency-associated peptide (Kueng et al., Anal. Biochem. 182: 16-19, 1989). Blockade of TGFβ activity is determined by a TMLC luciferase assay using mink lung epithelial cells expressing the TGFβ responsive portion of the PAI-1 promoter that drives firefly luciferase expression (Abe et al., Anal. Biochem. 216:276-284, 1994). Based on the hybridoma screening performed generally as described herein, the mouse hybridoma antibody ADWA-11 is selected for further evaluation.

Example 2: Humanization of Anti- α v β 8 Integrin Mouse Hybridoma Antibodies

The mouse hybridoma antibody ADWA-11, described in US Patent No. 9,969,804 and set forth in, for example , SEQ ID NOs: 20-33 and 71-76 herein, is humanized by grafting mouse CDR sequences into various human germline framework regions as listed in the table. 1, which include the germline light chain framework regions of IGKV2-28, IGKV2-30, IGKV4-1, IGKV1-39 (also referred to herein as DPK9) and IGKV3-11, as well as the germline heavy chain framework regions of IGHV3-7 (also referred to herein as DP54), IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69 and IGHV3-48 (see, for example, the IMGT database ).

The humanized antibodies, referred to herein as “Humanized ADWA-11,” comprise a set of six mouse CDR sequences set forth in SEQ ID NOs: 20-33 and 71-76 grafted into the germline IGKV1-39 light chain framework region (e.g. DPK9) and the germline heavy chain framework region of IGHV3-7 (eg, DP54). Other variants of the germline framework regions are also tested, as shown below in Tables 1.1 and 1.2.

Table 1.1

Humanized version SEQ ID NO ADWA11 IGHV1-46/IGKV1-39 VH: SEQ ID NO: 34;
VL: SEQ ID NO: 65 ADWA11 IGHV1-46/IGKV2-28 VH: SEQ ID NO: 34;
VL: SEQ ID NO: 62 ADWA11 IGHV1-46/IGKV3-11 VH: SEQ ID NO: 34;
VL: SEQ ID NO: 66 ADWA11 IGHV1-46/IGKV2-30 VH: SEQ ID NO: 34;
VL: SEQ ID NO: 63 ADWA11 IGHV1-46/IGKV4-1 VH: SEQ ID NO: 34;
VL: SEQ ID NO: 64 ADWA11 IGHV1-69/IGKV1-39 VH: SEQ ID NO: 37;
VL: SEQ ID NO: 65 ADWA11 IGHV1-69/IGKV2-28 VH: SEQ ID NO: 37;
VL: SEQ ID NO: 62 ADWA11 IGHV1-69/IGKV3-11 VH: SEQ ID NO: 37;
VL: SEQ ID NO: 66 ADWA11 IGHV1-69/IGKV2-30 VH: SEQ ID NO: 37;
VL: SEQ ID NO: 63 ADWA11 IGHV1-69/IGKV4-1 VH: SEQ ID NO: 37;
VL: SEQ ID NO: 64 ADWA11 IGHV3-30/IGKV1-39 VH: SEQ ID NO: 36;
VL: SEQ ID NO: 65 ADWA11 IGHV3-30/IGKV2-28 VH: SEQ ID NO: 36;
VL: SEQ ID NO: 62 ADWA11 IGHV3-30/IGKV3-11 VH: SEQ ID NO: 36;
VL: SEQ ID NO: 66 ADWA11 IGHV3-30/IGKV2-30 VH: SEQ ID NO: 36;
VL: SEQ ID NO: 63 ADWA11 IGHV3-30/IGKV4-1 VH: SEQ ID NO: 36;
VL: SEQ ID NO: 64 ADWA11 IGHV3-23/IGKV1-39 VH: SEQ ID NO: 35;
VL: SEQ ID NO: 65 ADWA11 IGHV3-23/IGKV2-28 VH: SEQ ID NO: 35;
VL: SEQ ID NO: 62 ADWA11 IGHV3-23/IGKV3-11 VH: SEQ ID NO: 35;
VL: SEQ ID NO: 66 ADWA11 IGHV3-23/IGKV2-30 VH: SEQ ID NO: 35;
VL: SEQ ID NO: 63 ADWA11 IGHV3-23/IGKV4-1 VH: SEQ ID NO: 35;
VL: SEQ ID NO: 64 ADWA11 IGHV3-48/IGKV1-39 VH: SEQ ID NO: 38;
VL: SEQ ID NO: 65 ADWA11 IGHV3-48/IGKV2-28 VH: SEQ ID NO: 38;
VL: SEQ ID NO: 62 ADWA11 IGHV3-48/IGKV3-11 VH: SEQ ID NO: 38;
VL: SEQ ID NO: 66 ADWA11 IGHV3-48/IGKV2-30 VH: SEQ ID NO: 38;
VL: SEQ ID NO: 63 ADWA11 IGHV3-48/IGKV4-1 VH: SEQ ID NO: 38;
VL: SEQ ID NO: 64 ADWA11 chimeric control

Table 1.2

Name Corresponding germ type SEQ ID NO: Subsequence ADWA11VH1 IGHV1-46 34 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMNWVRQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH2 IGHV3-23 35 EVQLLESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH3 IGHV3-30 36 QVQLVESGGGVVQPGRSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH4 IGHV1-69 37 QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYMNWVRQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VH5 IGHV3-48 38 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSS ADWA11VK1 IGKV2-28 62 DIVMTQSPLSLPVTPGEPASISCRSTKSLLHFNTYLFWYLQKPGQSPQLLIYYMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK2 IGKV2-30 63 DVVMTQSPLSLPVTLGQPASISCRSTKSLLHFNGNTYLFWFQQRPGQSPRRLIYYMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK3 IGKV4-1 64 DIVMTQSPDSLAVSLGERATINCRSTKSLLHFNGNTYLFWYQQKPGQPPKLLIYYMSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK4 IGKV1-39 65 DIQMTQSPSSLSSASVGDRVTITCRSTKSLLHFNTYLFWYQQKPGKAPKLLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11VK5 IGKV3-11 66 EIVLTQSPATLSLSPGERATLSCRSTKSLLHFNGNTYLFWYQQKPGQAPRLLIYYMSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCMQSLEYPFTFGQGTKVEIK

Sequence alignment comparing the amino acid sequences of the variable heavy chain region and variable light chain region of the mouse hybridoma ADWA-11 antibody (referred to as “mADWA-11 Hybridoma” and “mADWA11”), Humanized ADWA-11 antibody (“huADWA11-2.4”), and IGHV3- 07 and IGKV1-39 germline sequences are shown in FIG. 1A and 1B. The underlined amino acid residues are CDR sequences according to Kabat, and the amino acid residues in bold are the CDR sequences according to Chotia.

Example 3: Introduction of Framework Mutations into Humanized Anti- α v β 8 Integrin Antibodies

To improve the binding affinity of the humanized ADWA-11 antibody for αvβ8 integrin compared to the binding affinity of the mouse hybridoma ADWA11 antibody, several variants are generated having framework substitutions in the variable heavy chain (VH) region and the variable light chain (VL) region, as indicated in the table 2. The heavy chain residues described in this example are numbered as SEQ ID NO: 127. The light chain residues described in this example are numbered as SEQ ID NO: 128.

Table 2. Scaffold substitutions introduced into the humanized ADWA11 antibody

Option VH and VL areas ADWA11 Alternative name(s) Amino acid substitutions relative to SEQ ID NO: 127 (for heavy chain residues) and SEQ ID NO: 128 (for light chain residues) adwa_VH_1.1
(SEQ ID NO:88) ADWA11 VH01 T28N, F29I adwa_VH_1.2
(SEQ ID NO:89) ADWA11 VH02 T28N, F29I, R72A adwa_VH_1.3
(SEQ ID NO:90) ADWA11 VH03 T28N, F29I, R72A, A49G, L79A adwa_VH_1.4
(SEQ ID NO:91) ADWA11 VH04 T28N, F29I, R72A, N74T, A75S adwa_VH_1.5
(SEQ ID NO: 39) ADWA11 VH05
ADWA11 VH05_VK1 T28N, F29I, R72A, A49G, L79A, N74T, A75S adwa_VL_1.1
(SEQ ID NO: 47) ADWA11 VK01
ADWA11_VK01 (1) L46R adwa_VL_1.2
(SEQ ID NO:92) ADWA11 VK02 L46R, Y36F

Humanized ADWA11 antibody variants are created by combining different sets of framework substitutions listed in Table 2. Such combinations yield antibodies having VH ADWA11 VH01, ADWA11 VH02, ADWA11 VH03, ADWA11 VH04 or ADWA11 VH05 and VL of either ADWA11 VK01 or ADWA11 VK02.

Humanized ADWA11 antibody variants ADWA11 VH01/VK01, ADWA11 VH02/VK01, ADWA11 VH03/VK01, ADWA11 VH03/VK02 and ADWA11 VH05/VK01 are generated and evaluated as generally described herein.

The humanized ADWA11 antibody variant, designated ADWA11 VH01/VK01, includes the T28N and F29I substitutions in VH (SEQ ID NO:88) and the L46R substitution in VL (SEQ ID NO:47).

The humanized ADWA11 antibody variant ADWA11 VH02/VK01 includes T28N, F29I and R72A substitutions in VH (SEQ ID NO:89) and L46R substitutions in VL (SEQ ID NO:47).

The humanized ADWA11 antibody variant ADWA11 VH03/VK01 includes the T28N, F29I, R72A, A49G and L79A substitutions in VH (SEQ ID NO:90) and the L46R substitution in VL (SEQ ID NO:47).

The humanized ADWA11 antibody variant ADWA11 VH03/VK02 includes T28N, F29I, R72A, A49G and L79A substitutions in VH (SEQ ID NO:90) and L46R and Y36F substitutions in VL (SEQ ID NO:92).

The humanized ADWA11 antibody variant ADWA11 VH05/VK01 includes the T28N, F29I, R72A, A49G, L79A,N74T and A75S substitutions in VH (SEQ ID NO: 39) and the L46R substitution in VL (SEQ ID NO: 47).

The binding affinities of Fab ADWA11 VH01/VK01, ADWA11 VH02/VK01, ADWA11 VH03/VK01, ADWA11 VH03/VK02 and ADWA11 VH05/VK01 for αvβ8 integrin were determined as listed in Table 3. Additionally, IC50 values for inhibition of TGF-β activation were also determined for each antibody and they are listed in Table 3.

Table 3. Characterization of humanized ADWA11 antibody variants

Fab affinity TGFB IgG Efficiency Assay Antibody name ka kd KD(M) KD (pm) IC50 (pM) Mouse hybridoma antibody ADWA-11
VH: SEQ ID NO: 20
VL: SEQ ID NO: 21
Also called:
Hybridoma mADWA-11 9.22E+04 4.86E-05 5.36E-10 536 183
(n=3) Humanized variants (DP54/DPK9) ADWA11 VH01/VK01
VH: SEQ ID NO: 88
VL: SEQ ID NO: 47
Also called:
VH01/VK01 Fab 4.13E+04 2.51E-04 6.86E-09 6860 8473
(n=2) ADWA11 VH02/VK01
VH: SEQ ID NO: 89
VL: SEQ ID NO: 47
Also called:
VH02/VK01 Fab 1.30E+05 2.44E-04 1.89E-09 1890 1756
(n=2) ADWA11 VH03/VK01
VH: SEQ ID NO: 90
VL: SEQ ID NO: 47
Also called:
VH03/VK01 Fab 1.36E+05 <1E-05 7.35E-11 <100 138 ADWA11 VH03/VK02
VH: SEQ ID NO: 90
VL: SEQ ID NO: 92
Also called:
VH03/VK02 Fab 1.40E+05 5.23E-05 3.73E-10 370 223 ADWA11 VH05/VK01
VH: SEQ ID NO: 39
VL: SEQ ID NO: 47
Also called:
VH05-2_VK01;
VH05-2_VK01 original; and VH05/VK01 Fab 1.87E+05 <1E-05 5.00E-11 <100 148
(n=3)

As shown in Table 3, ADWA11 VH03/VK01, ADWA11 VH03/VK02 and ADWA11 VH05/VK01 each exhibit improved binding affinity for αvβ8 integrin compared to the mouse hybridoma antibody ADWA-11. ADWA11 VH03/VK01 (GBT) and ADWA11 VH05/VK01 each bind to αvβ8 integrin with a KD of less than 100 pM, which is at least 5-fold lower KD than the 536 pM KD determined for the mouse hybridoma antibody ADWA-11 . Additionally, ADWA11 VH03/VK01 and ADWA11 VH05/VK01 each exhibited a lower IC50 value for inhibition of TGF-β transactivation compared to the mouse hybridoma antibody ADWA-11.

It was then determined, as generally described herein, that ADWA11 VH05/VK01 also retains the activity, specificity, and cross-species reactivity of the mouse hybridoma antibody ADWA-11. Thus, this example demonstrates the improved binding affinity of the humanized ADWA-11 antibody for αvβ8 integrin compared to the binding affinity of the mouse hybridoma ADWA11 antibody.

Example 4: Optimization of Humanized ADWA11 Antibody Variants

The ability of the individual amino acid substitutions listed in Table 4 to improve the stability and/or reduce the immunogenicity of the humanized ADWA11 VH05/VK01 antibody was assessed. The heavy chain residues described in this example are numbered according to ADWA11 VH05 (SEQ ID NO: 39). The light chain residues described in this example are numbered according to ADWA11 VK01 (SEQ ID NO: 47).

Table 4. Selected amino acid substitutions

Light chain substitutions relative to ADWA11 VK01 (SEQ ID NO: 47) Heavy chain substitutions relative to ADWA11 VH05 (SEQ ID NO: 39) L30S K30A Y55A N55Q M56A N57Q N58S D61E A60Q P62A M94Q K63A L97Y F64V F101L F101W Q105G

As shown below, individual substitutions that include K30A, N55Q, N57Q, D61E, or P62A in the heavy chain variable region and L30S, M56A, N58S, M94Q, L97Y, or Q105G in the light chain variable region were found to retain the activity of the parent molecule (Fig. 4A-4B). Additionally, combinations of various individual amino acid substitutions listed in Table 5 are also evaluated and heavy chain sequences containing a double mutant comprising N55Q and D61E or a triple mutant comprising N55Q, D61E and F64V are found to retain the activity of the parent molecule as described hereinafter (FIG. 4C).

More specifically, ADWA11 VH05/VK01 variants are being created that include the combinations of amino acid substitutions listed in Table 5 and are referred to as ADWA11 2.1 (“2.1”), ADWA11 2.2 (“2.2”), ADWA11 2.3 (“2.3”) and ADWA112.4 ( "2.4"). The ADWA11 VH05-2/VK01, ADWA11 2.1 and ADWA11 2.4 variants, as described later herein, have been shown to have more favorable expression and activity profiles. This example demonstrates that individual amino acid substitutions improve the stability and/or reduce the immunogenicity of the humanized ADWA11 VH05/VK01 antibody.

Table 5. Combinations of amino acid substitutions

Clone name Alternative names Light chain substitutions relative to ADWA11 VK01 (SEQ ID NO: 47) Heavy Chain Replacements relative to ADWA11 VH05 (SEQ ID NO: 39) 2.1
VH: SEQ ID NO: 6
VL: SEQ ID NO: 67 ADWA11 2.1
VH05-2_VK01(2.1) L30S, M94Q, Q105G N55Q, D61E 2.2
VH: SEQ ID NO: 6
VL: SEQ ID NO: 68 ADWA11 2.2
VH05-2_VK01(2.2) L30S, L97Y, Q105G N55Q, D61E 2.3
VH: SEQ ID NO: 6
VL: SEQ ID NO: 69 ADWA11 2.3
VH05-2_VK01(2.3) L30S, M56S, M94Q, Q105G N55Q, D61E 2.4
VH: SEQ ID NO: 6
VL: SEQ ID NO: 7 ADWA11 2.4
VH05-2_VK01(2.4)
ADWA11 5-2 2.4 L30S, N58S, M94Q, Q105G N55Q, D61E 2.1 (F64V)
VH: SEQ ID NO: 93
VL: SEQ ID NO: 67 ADWA11 2.1 (F64V)
VH05-2(F64V)VK01(2.1) L30S, M94Q, Q105G N55Q, D61E, F64V 2.2 (F64V)
VH: SEQ ID NO: 93
VL: SEQ ID NO: 68 ADWA11 2.2 (F64V)
VH05-2(F64V)VK01(2.2) L30S, L97Y, Q105G N55Q, D61E, F64V 2.3 (F64V)
VH: SEQ ID NO: 93
VL: SEQ ID NO: 69 ADWA11 2.3 (F64V)
VH05-2(F64V)VK01(2.3) L30S, M56S, M94Q, Q105G N55Q, D61E, F64V 2.4 (F64V)
VH: SEQ ID NO: 93
VL: SEQ ID NO: 7 ADWA11 2.4 (F64V)
VH05-2(F64V)VK01(2.4) L30S, N58S, M94Q, Q105G N55Q, D61E, F64V

Example 5: Generation of anti- α v β 8 antibodies without effector function

Anti-αvβ8 integrin antibodies having reduced Fc-gamma receptor binding and reduced effector function (eg, reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and/or reduced complement-dependent cytotoxicity (CDC)) are created by subcloning the light chain variable region and heavy chain variable region of the invention and as listed in Table 1 in the immunoglobulin G (IgG) molecule.

To generate mouse antibodies without effector function, variable regions from the mouse hybridoma antibody ADWA-11 are subcloned into the Fc backbone of mouse IgG1, which contains the E233P, E318A, K320A and R322A single amino acid substitutions, as described in US Publication No. US2009/0155256. This antibody is referred to herein as ADWA-11_4mut and mIgG_4mut. Binding of ADWA-11_4mut to mouse avB8 was assessed using mouse astrocyte C8-S cells (ATCC), and blockade of TGFB activation was determined using C8-S cells in a TMLC co-culture luciferase assay.

To generate human antibodies lacking effector function, variable regions from the humanized antibody ADWA11 VH05VK01 are subcloned into a human IgG1 Fc backbone (e.g., as described herein) that contains L234A, L235A, and G237A single amino acid substitutions in the hinge region, such that the hinge region contains the amino acid sequence EPKSCDKTHTCPPCPAPE AAGA P (SEQ ID NO:126) instead of the wild type hinge region amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO:125). This human monoclonal antibody without effector function is designated herein as hIgG_VH05VK01.

Different characteristics of the light chain (SEQ ID NO: 123) and heavy chain (SEQ ID NO: 124) of hIgG_VH05VK01 are identified in the sequences below.

Light chain ADWA11 VK01

DIQ M TQSPSSLSASVGDRVTITC RSTKSLLHF N G NT YLF WYQQKPGKAPKRLI YY M SN LAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC MQSLEYPFT FGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 123)

Heavy chain ADWA11VH05

EVQLVESGGGLVQPGGSLRLSCAASGFNIK DYY M N WVRQAPGKGLEWVG WI DP DNG NT IYDPKFQ GRFTISADTSKNSAYLQ MN SLRAE DT AVYYCAR RLL M D YWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA G A PSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NST YRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV M HEA LHNHYTQKSLSLSPGK(SEQ ID NO: 124)

In the above light chain and heavy chain sequences of the humanized ADWA11VH05VK01, the CDR sequences are underlined; the N-linked glycosylation site of the consensus sequence is located at amino acid residues N295, S296 and T297 of the heavy chain; the potential cleavage site is located at amino acid residues D52 and P53 of the heavy chain; potential deamidation sites are located on amino acid residues N33, G34, N35 and T36 of the light chain and amino acid residues N57, T58, N77, S78, N84 and S85 of the heavy chain; the potential isomerization site is located on amino acid residues D90 and T91 of the heavy chain; the potential site for methionine oxidation is located on amino acid residues M4 and M56 of the light chain and M34, M102 and M426 of the heavy chain; the triple alanine mutant is located at amino acid residues A232 A233 and A235 of the heavy chain; and non-human residues outside the CDR are the amino acid residues R51 of the light chain and G49, A72, T74, S75 and A79 of the heavy chain. The residues in the heavy chain and light chain described in this example are numbered as SEQ ID NO: 124 and SEQ ID NO: 123, respectively.

Example 6: Evaluation of Anti- α v β 8 Integrin Antibodies Using ELISA

ELISA method

Biotinylated human αvβ8 integrin (50 μl of 0.6 μg/ml) was captured in a streptavidin-coated ELISA plate. After blocking and washing, antibodies of interest (eg, murine, chimeric, or humanized variants) are added at different dilutions to different wells and incubated at room temperature for 1 hour. After washing, the identifying antibody, anti-human IgG-HRP, is added and incubated for 1 hour at room temperature. After washing, enzyme substrate (TMB) is added to develop color for 10 minutes. The enzyme reaction is stopped by adding 0.16 M sulfuric acid, and the final signal intensity is measured at 450 nm.

results

Mouse hybridoma antibodies confirmed to bind human αvβ8 integrin (hαvβ8) are counter-screened for binding against the closely related human αvβ3 and human αvβ6 integrins to select mouse antibodies that bind specifically to hαvβ8. The mouse hybridoma antibody ADWA11 was specific for the hαvβ8 integrin and did not bind to the closely related integrins hαvβ3 or hαvβ6 (FIG. 2). However, the ADWA11 humanized antibody ADWA11 VH05/VK01 showed substantially improved affinity for αvβ8 integrin compared to the mouse hybridoma ADWA11 antibody (FIG. 3A-3B).

Anti-αvβ8 integrin Fab ADWA11 VH05/VK01 molecules having single or combinations of amino acid substitutions listed in Tables 4 and 5 were also assessed by ELISA for binding to hαvβ8 integrin (FIG. 4A-4C). As shown in FIG. 4A, Fabs having a K30A, N55Q, N57Q, D61E, P62A, or K63A single amino acid substitution in the heavy chain variable region retain binding affinity for hαvβ8. As shown in FIG. 4B, Fabs having a Y55A, A60Q, F101L or F101W single amino acid substitution in the light chain variable region exhibit reduced binding affinity for hαvβ8 compared to the parent antibody. As shown in FIG. 4C, Fabs having the combination of amino acid substitutions listed in Table 5 retain binding affinity for hαvβ8.

This example demonstrates that the ADWA11 humanized antibody ADWA11 VH05/VK01 shows substantially improved affinity for αvβ8 integrin compared to the mouse hybridoma antibody ADWA11, and some Fabs having individual amino acid substitutions retain binding for hαvβ8.

Example 7: Evaluation of Anti- α v β 8 Integrin Antibodies Using Biacore™

Way

Biotinylated recombinant αvβ8 integrin was captured on a streptavidin-coated Biacore™ chip (GE Healthcare Life Sciences) and the binding time response of Fab fragments was measured for a range of Fab concentrations. Generic background subtracted Biacore™ sensorgrams are obtained superimposed on the kinetic curve alignments.

More specifically, recombinant αvβ8 integrin (eg, R&D Systems) is labeled with biotin via primary amines and immobilized onto a Sensor Chip SA using a Biacore™ T200 instrument (GE Healthcare Life Sciences). Fab binding experiments were performed at 25°C using a flow rate of 30 μl/min in 0.01 M HEPES pH 7.4 buffer, 0.15 M NaCl, 1-2 mM MgCl ₂ and 0.005% v/v. surfactant P20 (HBS-P). Association is monitored for 5-10 minutes and dissociation for an additional 15-20 minutes for each Fab concentration. After each injection, the chip surfaces were restored with IgG wash buffer (Thermo Fisher Scientific). All data were analyzed using Biacore™ T200 Evaluation software. The data are background subtracted twice using an adjacent measurement cell coupled to streptavidin, without captured integrin, and with injections of buffer only. Obtain kinetic constants for at least three experiments and report them as averages.

results

Antibody binding to αvβ8 integrin from different species was assessed. Both mouse hybridoma ADWA-11 Fab and humanized ADWA-11 VH05-2/VK01(2.4) Fab were found to cross-react with αvβ8 integrin from other species. ADWA-11 Fab mouse hybridomas cross-react with human αvβ8 integrin, cynomolgus αvβ8 integrin, and mouse αvβ8 integrin. Humanized ADWA-11 VH05-2/VK01(2.4) Fabs cross-react with human αvβ8 integrin, cynomolgus αvβ8 integrin, and mouse αvβ8 integrin. Additionally, these antibodies are assessed for their ability to bind to the cognate integrins hαvβ3 or hαvβ6. These antibodies are specific for hαvβ8 and do not bind to the closely related integrins hαvβ3 or hαvβ6 in the Biacore assay, as shown in FIG. 5.

Comparison of the affinity measurements listed in Table 6 shows that the humanized ADWA-11 Fab has a lower KD for binding to both human αvβ8 integrin and mouse αvβ8 integrin, compared to the mouse hybridoma ADWA-11 Fab KD for binding to human αvβ8 integrin and αvβ8 mouse integrin. More specifically, the humanized ADWA-11 Fab has a KD of approximately 2.5-fold lower for binding to human αvβ8 integrin and a KD of approximately 3.5-fold lower for binding to mouse αvβ8 integrin, compared to the corresponding hybridoma ADWA-11 Fab KD values. mice. These data show an overall significant improvement in affinity for the humanized anti-αvβ8 antibody compared to the mouse hybridoma antibody (FIGS. 6A-6B and Table 6).

To evaluate the KD of the Fab ADWA112.4 monomer, also referred to herein as ADWA-11 VH05-2/VK01(2.4) and ADWA11 5-2 2.4, and the parent mouse IgG, recombinant human, cynomolgus, mouse, and rat αvβ8 were immobilized on the chip Biacore, and ka and kd for Fab are determined as generally described herein (Table 6). ADWA11 2.4 shows equivalent affinity for human, cynomolgus, mouse and rat αvβ8 with a KD <200 pM, however due to the very slow kd, accurate determination of the KD was not possible. The original mouse IgG showed equivalent KD affinities to human, cynomolgus and mouse αvβ8 (KD 489-536 pM) (rat not tested) (FIG. 6C).

Additional Biacore experiments refined the estimated KD values for ADWA11 2.4 to a KD of <100 pM for human and cynomolgus αvβ8 and 70.8 ± 19.9 pM (mean ± standard deviation) for mouse αvβ8.

Table 6. Affinity of α v β 8 integrin species according to Biacore (n ≥3)

Fab αvβ8 species ka (1/Ms) kd (1/s) KD Mouse hybridoma antibody ADWA-11
Also referred to herein as:
ADWA11 mouse Human 9.22E+04 4.86E-05 5.36E-7M mouse 1.03E+05 5.03E-05 4.89E-7M cynomolgus macaque 9.81E+04 4.97E-05 5.07E-7M ADWA11 5-2 2.4
Also called:
ADWA11 2.4
VH05-2_VK01(2.4) Human 1.61E+05 ≤3E-05 ≤2E-010 cynomolgus macaque 1.72E+05 ≤2E-05 ≤2E-010 mouse 2.57E+05 1.82E-05 ≤2E-010 rat 3.41E+05 ≤2E-05 ≤2E-010 Humanized ADWA11 Human 1.73E+05 3.61E-05 2.09E-7M mouse 1.77E+05 2.43E-05 1.37E-7M ADWA11 VH05-2_VK01
VH: SEQ ID NO: 39
VL: SEQ ID NO: 47
Also called:
ADWA11 VH05/VK01
VH05-2_VK01;
VH05-2_VK01 original; And
VH05/VK01 Fab Human 2.00E+05 2.37E-05 1.18E-10M mouse 2.20E+05 ≤2E-5 ≤1E-10M rat 1.94E+05 3.10E-05 1.60E-10M cynomolgus macaque 2.25E+05 ≤2E-5 ≤1E-10M

To assess the specificity of ADWA11 2.4, recombinant human αvβ6 and αvβ3 were immobilized on a Biacore chip and binding to the original humanized mouse ADWA11 2.4 mAb was determined. The parent hybridoma and humanized mAb do not bind αvβ3 or αvβ6, but binding to αvβ8 is observed in separate BIAcore experiments. The pan-integrin αV antibody is used to demonstrate the immobilization of αvβ3 or αvβ6 recombinant protein on a Biacore chip. Thus, this example also demonstrates the specificity of the humanized antibody for αvβ8.

Example 8: Evaluation of Anti- α v β 8 Integrin Antibodies in Cell Binding Assays

Methods

Cell binding experiments were performed with human glioblastoma U251 (Sigma) or C8-S (ATCC) cells cultured in MEM 10% hiFBS. Cells grown to 70-90% confluency are detached with 0.05% trypsin and washed twice with PBS containing 2% ABS.

For cell binding, HEK293-F cells overexpressing human αvβ3, αvβ5, αvβ6, αvβ8 were experimented with. HEK293-F cells transiently expressing full-length human integrin beta 3 (accession no. NP_000203.2), human integrin beta 5 (accession no. NP_002204.2), human integrin beta 6 (accession no. NP_000879.2) or human integrin beta 8 (accession no. access NP_002205.1) are obtained using proprietary vectors and protocols provided by the supplier. Cells were harvested after 4 days and integrin expression was analyzed. To characterize integrin expression αvβ3, αvβ5, αvβ6, αvβ8, 100,000 cells are incubated in the indicated commercially available or directly conjugated proprietary antibodies for 30 minutes, along with LIVE/DEAD fixable cell dye (Invitrogen) at 1:2000 to separate live cells. The cells are centrifuged for five minutes at 300 g. Cells are then washed twice with wash buffer (BSF + 0.2% ABS) to remove excess unbound antibodies and analyzed on a BD biosciences Fortessa flow cytometer.

Live cell binding protocol : 100,000 cells are incubated with a serial dilution of anti-αvβ8 or IgG1_3mut isotype human antibody for three to four hours on ice. The cells are centrifuged for five minutes at 300 g. The cells are then washed three times with wash buffer (BSF+0.2% ABS) to remove excess unbound antibodies. After thorough washing, cells are incubated with Fab'2 anti-human Fc-PE (Invitrogen) or Fab'2 goat anti-mouse-APC (Jackson Labs) at a dilution of 1:1000 along with LIVE/DEAD fixable cell dye (Invitrogen) at 1:2000 for separation of living cells. All contents are incubated for 30 minutes on ice. After washing, stained cells were analyzed on a BD biosciences Fortessa flow cytometer (live cell gated) using FlowJo analysis software. The mean fluorescence intensity (AFI) for different antibody concentrations is plotted for different antibodies.

Fixed Cell Binding Protocol : In some cases, cell binding experiments are performed with U251 human glioblastoma cells (Sigma) cultured in MEM or 10% hiFBS. Cells grown to 70-90% confluency are detached with 0.05% trypsin and washed twice with PBS containing 2% ABS. U251 (25,000 cells) was incubated with LIVE/DEAD fixed cell dye (Invitrogen) at 1:2000 to separate live cells. Cells are centrifuged for 5 minutes at 300 g and washed with wash buffer (FBS + 0.2% ABS), then fixed with 2% paraformaldehyde for 10 minutes at room temperature. Fixed cells are washed twice with wash buffer, then serial dilutions of the anti-αvβ8 antibody of interest are added for 1 hour at 37 degrees. Cells are washed three times with wash buffer to remove excess unbound antibodies. After thorough washing, Fab'2 anti-human Fc-PE (Invitrogen) or anti-human Kappa light chain-APC (Invitrogen) identifying antibody at a dilution of 1:1000 is added to the cells and incubated for 30 minutes on ice. After washing, stained cells were analyzed on a BD biosciences Fortessa flow cytometer (live cell gated) using FlowJo analysis software. The mean fluorescence intensity (AFI) for different antibody concentrations is plotted for different antibodies.

results

Fixed U251 cell binding data for ADWA11 VH05/VK01 Fabs having single amino acid substitutions in either the heavy chain variable region including F64V or the light chain variable region including L30S, M94Q, L97Y, F101L, F101W or Q105G, or a combination of amino acid substitutions , referred to as 2.1, 2.2, 2.3, 2.4, 2.1 (F64V), 2.2 (F64V), 2.3 (F64V) and 2.4 (F64V) in Table 5, are prepared and compared to the parent antibody (FIGS. 7A-7C).

U251 cell binding data were also obtained for antibodies mIgG_4mut and hIgG_VH05VK01 (also called ADWA11 2.4 and described further in Example 5) (FIG. 8). The apparent affinities of mIgG_4mut and hIgG_3mut_VH05VK01 for U251 cells are shown below in Table 7. These data demonstrate that hIgG_3mut_VH05VK01 was determined to have higher affinity compared to mIgG_4mut.

Table 7. Affinity values from U251 cell binding assay

ADWA11 antibody U251 app K _D (pM) mIgG_4mut 823 hIgG_3mut_VH05VK01 430

The apparent affinity of ADWA11 2.4 for U251 (human glioblastoma) or C8-S (mouse astrocyte) cells was also assessed in a cell binding assay according to the methods generally described herein. Briefly, cells were incubated with a serial dilution of ADWA11 2.4 for 4 hours on ice, then detected with a coupled anti-human-PE secondary antibody and analyzed by flow cytometry. ADWA11 2.4 exhibits saturable binding to cells expressing human αvβ8 and mouse αvβ8, with an average EC50 in the U251 (human αvβ8) cell binding assay of 126 pM with a standard deviation of plus/minus 34 pM (FIG. 9A; n=3).

In additional studies, the EC50 of ADWA11 VH05-2/VK01(2.4) binding to human U251 cells was determined to be 256 ± 115 pM (mean ± standard deviation of seven independent experiments), and binding to mouse C8-S cells was determined to be 145 ± 23. 7 pM (mean ± standard deviation of four independent experiments).

Cellular binding experiments with HEK293F transiently overexpressing β integrin family members demonstrate that ADWA11 2.4 specifically binds to cells expressing human αvβ8, but not αvβ3, αvβ5, or αvβ6 (FIG. 9B). Thus, ADWA11 2.4 exhibits improved performance compared to the parent mouse ADWA11 antibody, thereby suggesting that humanized ADWA11 2.4 is a potential improved therapeutic agent for humans.

Example 9: Evaluation of Anti- α v β 8 Integrin Antibodies in α v β 8 Induced TGF-β Activation Assays

Methods

The effect of anti-αvβ8 antibody on trans-activation of the TGFβ pathway was modified using U251-MG (Sigma) and Mv1Lu-SMAD-luciferase reporter cells. In some experiments, mouse C8-S astrocytes are used instead of U251 cells. Briefly, the mink lung epithelial cell line MvLu1 (ATCC) was transduced with Cignal SMAD (luc) reporter lentiviral particles (SABioscience) at a multiplicity of infection (MOI) of 50. Stable cell lines expressing the firefly luciferase SMAD construct were generated by culturing in complete growth medium (MEM). plus 10% fetal bovine serum (FCS) with L-glutamine + penicillin/streptomycin) with the addition of 2 μg/ml puromycin. For the experiment, U251 cells (5000 cells in 50 µl in MEM medium containing 2% purified on activated FBS) are added to each well of a TC-treated 96-well plate with a clear bottom and white walls, and incubated for 1 hour at 37°C. C. Serial dilutions of anti-αvβ8 antibodies, such as FAB antibodies, are prepared in MEM containing 2% purified FBS and added to seeded U251 cells, 25 μl per well. After an hour of incubation, Mv1Lu-SMAD-luciferase reporter cells are added (5000 cells/well in 25 μl) to each well and after 18 hours of incubation at 37°C, luciferase activity is measured using Bright Glo reagent (Promega) according to the protocol suggested by the manufacturer . Luminescence was measured using an Envision plate reader with an integration time of 1 s.

results

The inhibitory effect of different antibodies on αvβ8-induced transactivation of TGF-β U251 cells was monitored by assessing reduced luciferase activity (FIG. 10A-10F).

In FIG. 10A depicts a comparison of antibodies generated as described in Example 3 and includes a comparison between ADWA11 VH05/VK01 (also referred to as VH05-VK01 Fab) and the parent ADWA11 mouse hybridoma antibody (mFab). The EC50 value, measured through apparent affinity in the TGF-β transactivation assay, improves from 4.6 nM for parenteral ADWA11 mouse hybridoma antibody (mFab) to 1.3 nM for ADWA11 VH05/VK01 (also called VH05-VK01 Fab).

The effect of amino acid substitutions made in either the heavy chain variable region or the light chain variable region of ADWA11 VH05/VK01 on TGFβ U251 cell transactivation was also assessed (FIGS. 10B-10D). Fabs having an F101L or F101W single amino acid substitution in the light chain variable region exhibit reduced TGFβ transactivation compared to ADWA11 VH05/VK01 Fabs.

Comparison of the effect of humanized ADWA-11 IgG molecules on αvβ8-induced transactivation of TGF-β U251 cells was also monitored using a luciferase activity assay (FIG. 10E). These data indicate that the VH05/VK01-D61E and -N55Q-D61E mutants have comparable effects on TGFβ transactivation.

To evaluate the efficacy of ADWA11_VH05-2_VK01(2.4), alternatively referred to herein as ADWA11 2.4, a co-culture system with human and mouse cells that endogenously express αvβ8 was established with a TGFβ-luciferase sensing reporter cell system. Briefly, U251 (human glioblastoma) or C8-S (mouse astrocyte) cells were seeded with mink lung epithelial cells (Mv1Lu) stably transduced with Cignal SMAD reporter (luc) lentiviral particles (SABioscience) at a multiplicity of infection (MOI) of 50 (Mv1Lu- cells Smad). Mv1Lu-Smad cells respond to TGFβ generated by U251 or C8-S cells, and inhibition of αvβ8 function can be monitored through decreased luciferase activity. In FIG. 10F shows the effect of ADWA112.4 on TGFβ transactivation by U251 and C8-S cells compared to an isotype negative control antibody.

These data demonstrate that ADWA11 2.4 is a more effective inhibitor of αvβ8-induced TGF-β transactivation than other antibodies, including the mouse monoclonal antibody ADWA11. The IC50 for ADWA11 VH05-2/VK01(2.4) in the TGFβ transactivation assay with U251 cells was determined to be 199 ± 93.6 pM (mean ± standard deviation of five independent experiments).

Example 10: Evaluation of immunogenicity of anti- α v β 8 integrin antibodies

The functional significance of peptide binding to the Major Histocompatibility Complex (MHC) is assessed by T cell proliferation assay. CD4, a transmembrane glycoprotein expressed on T helper cells, recognizes peptides that bind MHC class II molecules on the surface of antigen presenting cells (APCs). This interaction causes proliferation of T helper cells, leading to an immune response. T cell proliferation is monitored through the decrease in fluorescence intensity of individual cells containing the dye carboxyfluorescein succinimidyl ester (CFSE). ProImmune's REVEAL® Immunogenicity System T-cell assay uses flow cytometry techniques to analyze the division of CFSE-labeled cells. PMBC from different donors are incubated with CFSE to obtain intracellular fluorescent conjugates. The fluorescence intensity of CFSE decreases by half with each subsequent cell division, allowing cell proliferation to be measured. This robust and reproducible CFSE-labeled T cell assay is used to identify potential CD4 T cell epitopes on MHCII presented peptides. Peptide controls derived from influenza/tetanus and purified protein derivative of tuberculin (PPD) are used as positive controls for cell proliferation.

Twenty-nine peptides spanning the CDRs were tested with PBMCs obtained from 51 T cell proliferation donors (Table 1, SEQ ID NO: 94-122). CFSE-labeled PBMCs are incubated with test peptides for seven days, and the extent of CD4+ T cell proliferation is monitored by flow cytometry. The number of responders for a peptide derived from the optimized molecule is compared with the number of responders for corresponding germline peptide sequences.

Reference antigens containing known MHC class II epitopes are used in this study.

Purified protein derivative of tuberculin (PPD) is a derivative of Mycobacterium tuberculosis and is used at a final assay concentration of 5 μg/ml. It is expected that approximately 70-100% of PBMC donors will respond to this protein as a result of previous vaccination, i.e. through an immune memory response.

Snail limpet hemocyanin (KLH) is a recognized and effective intact protein immunogen used at a final concentration of 0.25 mg/mL in the assay. Typically, 50-80% of donors can be expected to respond to this protein, presumably driven by an intact immune response.

A synthetic HA peptide is derived from influenza A hemagglutinin (PKYVKQNTLKLAT, residues 307-3192; SEQ ID NO: 129) and used at a final assay concentration of 5 μM. It is expected to elicit a response in up to 50% of donors.

Synthetic TT peptide (AQYIKANSKFIGITEL (SEQ ID NO:130), TET 830 modified/T-helper epitope from tetanus toxin) is a universal T-cell epitope of human tetanus toxin, and is used as a helper peptide in vaccination. It is used at a final assay concentration of 5 µM and is expected to respond in up to 45% of donors.

The F64V substitution (SEQ ID NO: 105) in the heavy chain was found to enhance immunogenicity. Eleven of 51 donor PBMCs responded in the CD4+ T cell proliferation assay, and none of the donors was sensitive to the corresponding germline sequence. Other substitutions including Q105G (SEQ ID NO: 111), L30S (SEQ ID NO: 106), M94Q (SEQ ID NO: 107) and N58S (SEQ ID NO: 113) were found to reduce immunogenicity, all to varying degrees. degrees, as shown in Table 8. The percentage of responders for different CDR peptides compared to the positive control peptides is given in FIG. eleven.

Table 8

Number of responders ^# for peptides corresponding to different substitutions Replacement Original After replacement F64V 0 eleven Q105G 6 3 L30S 6 0 M94Q 6 0 N58S 5 3

^# number of PBMC donors responding to CD4+ T cell proliferation assay

These data demonstrate that certain antibodies of the invention, including ADWA11 2.4, exhibit reduced T cell responses compared to the parent ADWA11 mouse mAb. These results suggest that ADWA11 2.4 is an improved potential therapeutic agent for humans compared to the parent mouse antibody.

Example 11: α v β 8 Inhibition Improves Efficacy of Anti-PD-1 Therapy in EMT6 Tumor Model

Methods

In this study, 3x10 ⁵ EMT6 (ATCC) cells were implanted into the quarter mammary fat pad or subcutaneously in Balb/c mice (Charles River Laboratory). Mice are randomly assigned to treatment groups when their tumors reach an average size of 50 mm ³ and then begin treatment. For treatment, mice receive a 10 mg/kg dose of the indicated 2A3_rat IgG antibody (“2A3”; BioXcell), anti-PD-1 antibody (“PD1”; clone RMP1-14, BioXcell), 2B8_mIgG1_4mut (“2B8”), or ADWA11_mIgG1_4mut ( ADWA11) by intravenous injection every four days for a total of three injections. Tumors are measured in two directions to track growth, where volume (V) = ½ L × W ² , and L (length) is defined as the largest diameter of the tumor and W (width) perpendicular to L. Tumor measurements are recorded 2-3 times per week until the end research.

results

Anti-αvβ8 (ADWA11) in combination with anti-PD1 therapy (ADA11+PD1) synergistically and significantly reduced tumor growth and improved survival compared with groups treated with anti-PD1 or anti-αvβ8 monotherapy (Figures 12A and 12B).

In the 10 mg/kg ADWA11 monotherapy group, treatment produced 47.1% tumor growth inhibition (TGI) at day 13 of the study, however, the TGI was transient and none of the mice reached the end of the study (0% survival at day 51). Anti-PD1 monotherapy produced a 15% TRI rate at day 14 of the study, and none of the mice reached the end of the study (0% survival at day 51). In contrast, ADWA11 (10 mg/kg dose per group) in combination with an anti-PD-1 antibody produced a 90.0% TRI on day 14 of the study, and 60% of mice reached the end of the study (60% survival at 51 days).

These results demonstrate for the first time that ADWA11 antibodies, including ADWA11 2.4, provide a synergistic therapeutic effect when combined with a PD-1 inhibitor, such as an anti-PD-1 antibody. These data suggest that ADWA11 2.4 is a potential human therapeutic agent that may provide a synergistic antitumor response when combined with a PD-1 inhibitor.

Example 12: α v β 8 Inhibition Improves the Efficacy of 4-1BB and Anti-CTLA4 Therapy in an EMT6 Model

Methods

In this EMT6 tumor efficacy study, 1x10 ⁶ EMT6 (ATCC) cells were implanted into the quarter mammary fat pad of Balb/c mice (Charles River Laboratory). Mice are randomly assigned to treatment groups when their tumors reach an average size of 100 mm ³ and then begin treatment. Mice receive i.v. dose 4-1BB (MAB9371 R&D Systems, 1 mg/kg Day 0 and Day 4), anti-CTLA4 (clone 9D9 BioXcell, 10 mg/kg Day 0, Day 4 and Day 8), anti-αvβ8 (ADWA11, 10 mg /kg Day 0, Day 4 and Day 8), 2B8_mIgG_4mut (10 mg/kg, Day 0, Day 4 and Day 8) or 2A3_rat IgG (BioXcell, 10 mg/kg Day 0, Day 4 and Day 8). Tumors are measured in two directions to track growth, where volume (V) = ½ L × W ² , and L (length) is defined as the largest diameter of the tumor and W (width) perpendicular to L. Tumor measurements are recorded 2-3 times per week until the end research.

Immunohistochemistry (IHC) analysis : 4 mm thick formalin-fixed paraffin-embedded (FFPE) tumor tissue sections for CD8, CD45 and Granzyme B expression using custom protocols and Leica Bond-max automated IHC stain. Images are acquired on a Leica/Aperio AT2 digital whole-mount scanner using 20× magnification. Images are analyzed using custom algorithms created in Visiopharm 7.2 software and optimized for each target of interest. Cell counts are performed on viable tissue and normalized to the area of viable tissue. Cell density is calculated using the following equation: Cells/µm2 = (#Positive Cells/Viable Tumor Area (µm2))*1 x 106. Percentage density of the object is calculated using the following equation: Staining Area (µm2)/Viable Tumor Area (µm2) .

results

Anti-αvβ8 (ADWA11) in combination with anti-4-1BB or anti-CTLA4 significantly and synergistically reduced tumor growth and improved survival compared with groups treated with anti-4-1BB, anti-CTLA4 or anti-αvβ8 monotherapy (FIG. 13).

Changes in tumor infiltrating cell density after treatment with anti-αvβ8 antibody were assessed (FIG. 15). Lymphocyte numbers were counted in the EMT6 tumor model using IHC staining density analysis for CD45 (total lymphocytes and myeloid cells), CD3 (total T cells), CD4 T cells, CD8 T cells, and Granzyme B (activated CD8 and NK cells). These data demonstrate that anti-αvβ8 monotherapy increases the numbers of total CD45+ cells, CD4+ T cells and CD8+ T cells, and causes a very significant increase in the density of Granzyme B-expressing cells (n=10 for each group).

The number of tumor lymphocytes in tumor tissue was analyzed on Day 11 (antibody treatment on Days 0, 3, 6, 9) in mice treated with isotype control or ADWA11 (2.4) (FIG. 15). Treatment with ADWA11 (2.4) increases the density of total leukocytes (CD45+, 1540±558 to 2470±407), CD8 T cells (CD8+, 76.3±62.7 to 170±74.2) and cytotoxic cells (% Granzyme B density, 11.8±11.0 to 106±35.1) in the tumor microenvironment (mean CD45+ cell count, mean CD8+ cell count, or mean % Granzyme B staining area per ^mm2 ± standard deviation in ADWA11 isotype treatment (2.4)).

These results demonstrate for the first time that ADWA11 antibodies, including ADWA11 2.4, provide a synergistic therapeutic effect compared with a 4-1BB agonist, such as an anti-4-1BB antibody. These data suggest that ADWA11 2.4 is a potential human therapeutic agent that may provide a synergistic antitumor response when combined with a 4-1BB agonist.

These results also demonstrate for the first time that ADWA11 antibodies, including ADWA11 2.4, provide a synergistic therapeutic effect when combined with a CTLA4 inhibitor, such as an anti-CTLA4 antibody. These data suggest that ADWA11 2.4 is a potential human therapeutic agent that may provide a synergistic antitumor response when combined with a CTLA4 inhibitor.

Example 13: α v β 8 Inhibition Improves the Efficacy of Radiation Therapy in the CT26 Tumor Model

Methods

CT26 Efficiency Assay : 4x10 ⁵ CT26 (ATCC) cells were implanted subcutaneously in the flank of Balb/c mice (Charles River Laboratory). Mice are randomly assigned to treatment groups when their tumors reach an average size of 100 mm ³ and then begin treatment. Mice receive 10 mg/kg i.v. dose every 4 days, 3 doses total, 2B8_mIgG1_4mut (self) isotype control or ADWA11_mIgG1_4mut and one dose of 5 Gy tumor targeted radiation on Day 5 after the first dose. Tumors are measured in two directions to track growth, where volume (V) = ½ L × W ² , and L (length) is defined as the largest diameter of the tumor and W (width) perpendicular to L. Tumor measurements are recorded 2-3 times per week until the end research.

qPCR Gene Expression Analysis : Tumor tissue is collected and 30 mg of tissue is homogenized in 900 µl of lysis buffer supplied in the RNeasy Plus Mini Kit using an Omin Bead Ruptor. RNA from homogenized tumor samples was isolated using the RNeasy Plus Mini Kit and protocols recommended by the supplier. cDNA was synthesized using 2 μg of total RNA and a high-throughput cDNA reverse transcription kit using protocols recommended by the supplier. Gene expression was analyzed using 50 ng cDNA and gene-specific linear degradable probe primers, TaqMan Universal Master Mix II and protocols recommended by the supplier. The ViiA7 qRT-PCR system is used for qPCR research. Threshold cycles (CT) for each sample are analyzed using the recommended comparative CT method, and expression of target genes is shown as fold change in the treatment group compared to the isotype control group. An unpaired two-tailed Student's t test was used to compare the treatment group with the isotype control group with statistical significance expressed as <0.05.

Immunohistochemistry (IHC) analysis : 4 mm thick formalin-fixed paraffin-embedded (FFPE) tumor tissue sections for CD8, CD45 and Granzyme B expression using custom protocols and Leica Bond-max automated IHC stain. Images are acquired on a Leica/Aperio AT2 digital whole-mount scanner using 20× magnification. Images are analyzed using custom algorithms created in Visiopharm 7.2 software and optimized for each target of interest. Cell counts are performed on viable tissue and normalized to the area of viable tissue. Cell density is calculated using the following equation: Cells/μm2 = (#Positive Cells/Viable Tumor Area (μm2))*1 x 106.

results

Anti-αvβ8 antibody (ADWA11) in combination with radiation therapy significantly reduced tumor growth and improved survival compared with radiation therapy alone (FIG. 14A).

In the CT26 studies, ADWA11 antibody (anti-ITGαVβ8) monotherapy produced a 64.3% TRI rate at Day 19 of the study, but the response was transient and only 1 of 10 mice reached the end of the study (10% survival, Day 57). Radiation therapy alone (5 gray (Gy) dose on Day 5) produced 57.7% tumor growth inhibition (Day 18) and 1 in 20 mice reached the end of the study (5% survival, Day 57). In contrast, a comparison of ADWA11 treatment in combination with radiation therapy resulted in 87.7% tumor growth inhibition (Day 19, 10 mg/kg ADWA11 dose and 5 Gy radiation therapy per group) and 9 of 19 mice reached the end of the study (47.4% survival , Day 57).

To examine the effect of ADWA11 on tumor lymphocyte numbers in the CT26 tumor model, tumor tissue was collected on Day 12 (antibody treatment on Days 0, 4, 8) from mice treated with isotype control or ADWA11 VH05-2/VK01 (anti-ITGαVβ8) and analyzed for lymphocyte markers using IHC (FIG. 14B, top panel). ADWA11 VH05-2/VK01 treatment increases total CD45+ leukocyte density; Isotype: 367±128, ADWA11 VH05-2/VK01: 695±94.8 (mean number of cells per mm ² ± standard deviation). CD8+ T cell; Isotype: 173±79.3, ADWA11 VH05-2/VK01: 374±80.4 (Mean number of cells per mm ² ± standard deviation). Granzyme B expressing cytotoxic cells, Isotype: 264±65.5, ADWA11 VH05-2/VK01: 514±91.7 (Mean number of cells per mm ² ± standard deviation) in the tumor microenvironment. Additionally, anti-ADWA11 treatment in combination with radiation therapy increased the mRNA expression levels of CD45 (3.86±0.979), CD8a (5.45±3.53), Granzyme B (4.21±1.02) and IFNγ (5 .53±2.13) in the tumor microenvironment (Fold increase ± standard deviation to Isotype treatment group) (FIG. 14B, bottom panel).

These studies demonstrate that anti-ITGαVβ8 (ADWA11_mIgG1_4mut) treatment increases the abundance of activated lymphocytes in the tumor microenvironment and, in combination with targeted tumor irradiation, is effective for tumor regression and long-term survival.

These results also demonstrate for the first time that ADWA11 antibodies, including ADWA11 2.4, provide a synergistic therapeutic effect when combined with radiation therapy. These data suggest that ADWA11 2.4 is a potential human therapeutic agent that may provide a synergistic antitumor response when combined with radiation therapy.

Example 14: Evaluation of α v β 8 Integrin as a Novel Suppressor of Tumor Immunity and Target for Antitumor Immunotherapy

In this study, an effective αvβ8-blocking monoclonal antibody (ADWA-11), which was generated by immunizing Itgb8 knockout mice with recombinant αvβ8, is used to test whether inhibition of this integrin can promote antitumor immunity.

In non-tumor tissues, αvβ8 integrin is expressed on neuroepithelium, fibroblasts, dendritic cells and T cells and can activate latent transforming growth factor β (TGFβ), an important immunomodulator. We show herein that in carcinomas, myeloid cells express αvβ8 integrin and that an effective αvβ8 blocking monoclonal antibody (ADWA-11) causes tumor growth suppression or complete tumor regression in syngeneic models of squamous cell carcinoma, breast cancer, and colon cancer, particularly when combined with other immunomodulators (anti-PD1, anti-CTLA-4 or 4-1BB) or radiation therapy. ADWA-11 treatment increases tumor infiltration and Granzyme B CD8+ T cell expression and improves the ratio of proinflammatory to suppressive tumor-associated macrophages. Most human tumors express ITGB8 mRNA, and as shown herein, high levels of surface expression of αvβ8 are observed on monocytes, macrophages, and dendritic cell subsets in biopsies of human ovarian carcinoma and renal cell carcinoma. These data identify αvβ8 integrin as a promising new target for anticancer immunotherapy.

Effective synergistic combination of immunotherapy with ADWA-11 and anti-PD-1 in a squamous cell carcinoma model

The effect of ADWA11, alone or in combination with an anti-PD-1 antibody, was studied in a syngeneic tumor model of verified squamous cell carcinoma (CCK168 cells) (FIG. 16A and FIG. 28). CCK168 cells, a chemically induced squamous cell carcinoma cell line derived from FVB mice, were injected subcutaneously into the dorsolateral right flank of syngeneic wild-type FVB mice at a dose of 1.5 x 10 ⁴ cells/mouse.

Tumors are allowed to grow for more than 14 days. Mice selected for the experiment had a tumor size of at least 5 mm in diameter and were optimally allocated based on tumor size into different treatment groups using the Studylog program. Mice were weighed and tumor size was measured every other day throughout the study using a monitorable digital caliper (Fisher Scientific, model #14-648-17). Mice are sacrificed when tumor size reaches or exceeds 2000 mm ³ or significant ulceration develops at the tumor site.

Mice are treated with ADWA11 hybridoma antibody or isotype-matched control antibodies on days 0 and 7 and anti-PD-1 mouse antibody (RMP1-14, BioXcell) or its isotype-matched control antibody on days 0, 4, and 8 (day 0 is the first day of therapy ). The appropriate antibody for each group and isotype control antibodies are administered by intraperitoneal injection at doses of 10 mg/kg for each antibody, ADWA11, anti-PD-1 antibody (RMP1-14, BioXcell), control antibody ADWA-21 (for ADWA11), and control 2A3 (BioXcell). Control ADWA-21 binds human but not mouse integrin-β8.

CCK168 tumors exhibit minimal responses to anti-PD1 antibody, but five of ten mice treated with ADWA11 monotherapy show tumor regression (FIGS. 16B-16C). Combination therapy with ADWA11 and anti-PD1 antibody causes complete regression in eight of ten tumors and a significant increase in overall survival when mice are treated with ADWA11 hybridoma and anti-PD1 antibody (FIG. 16B) or ADWA11_4mut_mIgG1 and anti-PD1 antibody (FIG. 28) .

Surviving mice were followed up to two years after tumor regression, and none showed evidence of subsequent tumor regrowth. Thirteen mice from two replicate experiments with complete regression after combination therapy and 3 mice with complete regression after ADWA11 monotherapy were rechallenged once or twice with CCK168 cells, and none showed tumor growth, indicating the development of long-term immunity to the tumor .

These results demonstrate for the first time that ADWA11 antibodies, including ADWA11 2.4, provide a synergistic therapeutic effect when combined with a PD-1 inhibitor, such as an anti-PD-1 antibody. These data suggest that ADWA11 2.4 is a potential human therapeutic agent that may provide a synergistic therapeutic antitumor response when combined with a PD-1 inhibitor.

Surface integrin α v β 8 is present on myeloid cells in the tumor microenvironment

Flow cytometry is used to identify which tumor cell types express β8 integrin, presumably expressed as an αvβ8 heterodimer (FIG. 17A and FIG. 17B). αvβ8 expression is readily detected on more than 80% of CD45+CD11b+F4/80+CD64+ macrophages and less than 20% CD45+CD11b+F4/80-CD64-CD11c+MHCII ^high dendritic cells in CCK168 tumors (Figure 17B). αvβ8 is expressed at similar levels in each of the macrophage subpopulations examined, including early infiltrating (proinflammatory) Ly6C+ cells and immunosuppressive CD206+ cells (FIG. 17B). αvβ8 is also expressed on CCK168 tumor cells in vitro (FIG. 18B). Low levels of αvβ8 expression on CD45 tumor and stromal cells in vivo were also found (FIG. 18A). No αvβ8 expression was found on intratumoral T cells (FIG. 20).

Animals

All animal studies were conducted in accordance with approved protocols from the University of California, San Francisco, Institutional Animal Care and Use Committee of Pfizer, Inc. Institutional Animal Care and Use Committee. The wild-type FVB/N mice used were either purchased from Jackson Laboratories (The Jackson Laboratories, stock #001800) or obtained from our own breeding colony obtained from this stock. Wild-type Balb/c mice were purchased from Charles River Laboratories (Charles River Laboratories, strain code 028).

Treatment of human tumor

Informed consent was obtained from all subjects for all human samples, and the work was performed in accordance with Institutional Review Board (IRB) approval. Fresh tissues are collected and processed according to the workflow design of UCSF Immunoprofiler, a translational platform designed and optimized for profiling immune subsets within cancers. Briefly, tissue is collected from the operating room and transported to the laboratory within 4 hours of collection. The tissue is extensively minced (<1 mm pieces) and enzymatically digested (3 mg/ml collagenase A, 50 U/ml DNase I) according to established standard operating procedures. Immune populations are subjected to multiplex flow cytometry (>60 colors) to analyze the proportionality and mean fluorescence intensity of known subsets and their αvβ8 expression. All antibodies were purchased from BD Pharmingen, eBioscience, Invitrogen, or BioLegend. Anti-αvβ8 antibody was prepared as previously described. All flow cytometry, including cell sorting, is performed on a custom-ordered BD FACSAria Fusion flow cytometer. Flow cytometry data analysis is performed using FlowJo.

Flow cytometry protocol

Subcutaneous tumors are isolated from mice using scissors and blunt dissection. Tumors are placed in a Petri dish with a digestion cocktail of Collagenase XI (Sigma C9407) 2 mg/ml, Hyaluronidase (Sigma H3506) 0.5 mg/ml and DNase (Sigma DN25) 0.1 mg/ml, obtained in C10 medium ( RPMI 1640, Hepes 1%, penicillin/streptomycin 1X, fetal bovine serum 10%, sodium pyruvate 1 mM, non-essential amino acids 1X and beta-mercaptoethanol 0.45%). Tumors are crushed with sterile scissors. The resulting cell suspension is transferred into 50 ml conical tubes (Fisher Scientific #14-432-22), and the Petri dish used to grind the tumor is washed with 2 ml of C10 medium to catch the remaining cells. Cells are incubated in a shaker at 255 rpm for 45 minutes at 37C. After incubation, 15 ml of C10 medium is added to the digested tumor cells and shaken gently for 15 seconds. The cell suspension is passed through a 100 µm mesh filter (Falcon® #352360) into a clean 50 ml conical tube. Cells are pelleted by centrifugation for 5 minutes at 200 g at 4°C and reconstituted in PBS. Cell counts were performed using a Countless II FL hemocytometer (Life Technologies).

Isolated preparations of individual cells are used for surface and intracellular staining. After counting, 10 x 10 ⁶ cells are transferred to each well of a 96-well v-shape plate for staining. Live/dead stain with Ghost Dye™ Violet 510 (TONBO bioscience #13-0870) at 1:1000 for 20 minutes at 4°C. Fc receptor and non-specific binding were blocked with anti-CD16/30 (eBioscience #14061) for 10 minutes at 4°C. Surface staining is carried out for 20 minutes at 4°C. For intracellular staining, cells are incubated in Fix/Perm buffer (eBioscience #88-8824) for 20 minutes at room temperature followed by intracellular cytokine staining with antibody cocktails for 20 minutes at 4°C. After completion of staining, cells were transferred to flow cytometry buffer (FBS with 2% FCS, penicillin/streptomycin/glutamate, EDTA 2 mM) for analysis.

Antibodies used for T cell staining experiments: ICOS FITC (eBioscience#11-9949-80), CD25 AF780 (eBioscience#47-0251-82), CD45.1 AF700 (BioLegend#110723), CD8 BV605 (BioLegend#100743 ), CD4 BV650 (BioLegend#100546), Ki-67 PE-Cy7 (BD Biosciences#561283), CTLA4 PE (BD Biosciences#553720) and FoxP3 PB-e450 (eBiosciences#48-5773-82).

Antibodies used for intracellular cytokine staining experiments: CD3 APC (eBioscience#17-0032-82), NK1.1 APC-AF780 (eBioscience#47-5941-80), CD45.1 AF700 (BioLegend#110723), CD4 BV650 ( BioLegend#104729), CD8 BV605 (BioLegend#100546), IFN-γ FITC (eBioscience#11-7311-82), IL-17A PE-Cy7 (BioLegend#506921), Granzyme-B PE (eBioscience#12-8898- 82), FasL PerCP-eFluor710 (eBioscience#46-5911-82) and FoxP3 PB-e450 (eBioscience#48-5773-82).

Antibodies used for experiments of myeloid cell staining: LY6G BV785 (Biolegend#127645), SIGLECF BV785 (BD BIOSCIENCES#740956), CD90.2 BV785 (BIOLEGEND#10533), B220 BV785 (BIOLEGEND#10 3246), CD45.1 AF700 (BioLeGEND #110724), CD11b AF780 (eBioscience#47-0112-82), CD206 PerCP-Cy5.5 (BioLegend#141716), F4/80 PE (BioLegend#123109), CD11c BV650 (BioLegend#117339), Ly6C BV605 (BioLegend #128035), MHC-II PB-e450 (eBioscience#48-5321), CD24 PE-Cy7 (BioLegend#101822), CD103 FITC (eBioscience#11-1031-82), ADWA11 APC (conjugated in our laboratory) and CD64 FITC (BioLegend#139316).

Antibodies used for NK cell staining experiments: CD45.1 AF700 (BioLegend#110724), CD3 APC (eBioscience#17-0032-82), NK1.1 APC-AF780 (eBioscience#47-5941-80), CD314- NKG2D PE (eBioscience#12-5882-82), CD226-DNAM-1 PerCP-Cy5.5 (BioLegend#128814), CD335-NKp46 FITC (eBioscience#11-3351-82), CD107a-LAMP-1 PE-Cy7 (BioLegend#121620) and CD49b eFluor450 (eBioscience#48-5971-82).

To stimulate cells, cells are stimulated until cell surface and intracellular staining occurs. Approximately 3x10 ⁶ cells in 200 μl C10 medium per well are incubated in round bottom 96 well plates in a tissue culture incubator in 5% CO ₂ at 37°C. A stimulation cocktail (Inomycin, PMA, Brefeldin-A and Monensin 500x Tombo stimulation cocktail #TNB4975-UL100) is added to the cells, which are incubated in a tissue culture incubator in 5% CO ₂ at 37°C for 4 hours. Cells were transferred to v-bottom wells for staining as described above. Flow cytometry was performed using BD LSRFortessa™ (BD Biosciences) and analyzed using FlowJo™ (Tree Star Inc.).

ADWA11 increases tumor infiltration and improves the differentiation of cytotoxic CD8+ T cells and increases the ratio of inflammatory monocytes to suppressive macrophages

The effect of ADWA11 (with or without anti-PD1) on the nature of the immune infiltrate has also been characterized (Thomas et al., Cancer Cell 8:369-380, 2005; Wu et al., Cancer Immunol. Res. 2:487-500, 2014) . Although anti-PD1 treatment in this model had no effect on the total number of tumor CD8+ T cells, ADWA11 treatment significantly increased CD8 T cell infiltration, an effect that was most evident in immunostaining of excised tumors (Figures 19A-19B). ADWA11 also significantly increased the proportion of CD8+ T cells that express Granzyme B (FIG. 19C), while anti-PD1 treatment had no effect. Inflammatory monocytes also play a role in tumor evasion from the host immunological response. Ly6C is expressed on inflammatory monocytes, and Ly6C expression is attenuated in tumor-associated macrophages with suppressive properties in the tumor microenvironment (Franklin et al., Science 344:921-925, 2014; Movahedi et al., Cancer Res. 70:5728-5739). Additionally, classification of the myeloid population shows that ADWA11 treatment specifically increases the accumulation of CD45+CD11b+CD11c-Ly6G-Ly6C ^high CD206 ^low inflammatory macrophages (FIG. 19D) (Ostuni et al., Trends Immunol. 36:229-239, 2015; Noy et al., Immunity 41:49-61, 2014). Neither treatment had a significant effect on the number of CD4+ T cells, CD4+ FoxP3+ regulatory T cells, or interferon-γ expression by T cell subsets at the time points analyzed, and we were unable to identify significant IL-17 expression in any T cell subsets (data not shown).

For immunostaining, tumors were collected and fixed in 4% paraformaldehyde at 4°C overnight. Fixed tumors are immersed in 30% sucrose solution at 4°C overnight and embedded in OCT compound (Tissue Tek® #4583) and cryosectioned at 15 μm. Frozen sections were stained using previously described protocols (Henderson et al., Nat. Med. 19:1617–1624, 2013; Rock et al., Proc. Natl. Acad. Sci . USA 108:E1475–1483, 2011). Briefly, cryosections were permeabilized and blocked with 0.3% Triton X-100 and 3% ABS in PBS. Sections were incubated with primary antibodies overnight at room temperature, then with fluorophore-conjugated primary and secondary antibodies, and then prepared with Prolong Gold (Invitrogen).

Antibodies used for immunostaining: rat anti-F4/80 (Alexa Fluor 647-conjugated, Serotec, clone C1, 1:100), rabbit anti-phospho-Smad3 (Epitomics, 1880-1; 1:100), anti-CD8 rats (Alexa Fluor 488- or 594-conjugated, Biolegend, clone 53-6.7, 1:100). Alexa Fluor 488-, 555-, 647-conjugated anti-rabbit and anti-rat donkey (Invitrogen). Confocal microscopy was performed using Zeiss LSM780 and LSM5 Pascal microscopes.

All quantifications are performed using high-resolution confocal images representing a thin (1 air unit; ~1 μm) optical section of the sample. Images were analyzed using ImageJ software. Each group contained samples from at least 5 control and 5 treated (ADWA11) mice. Four images (field size 425.10 x 425.10 μm) of each tissue section were acquired randomly using the same confocal settings. Images are placed with equal borders, then the area covered by myofibroblast or phospho-Smad3 spots is calculated.

Beneficial synergistic effects of combined ADWA11 and anti-PD1 therapy are abolished by CD8+ T cell depletion

Because the most potent effects of ADWA11 therapy were observed on CD8+ T cells, we herein sought to determine whether these cells drive the antitumor effects of ADWA11. In mice bearing the CCK168 tumor, cytotoxic T cells are eliminated using anti-CD-8a (Bio X Cell® BE0004-1 clone 53-6.72) antibody or control antibody at a dose of 10 mg/kg, which is administered intraperitoneally over 24 hours before each administration of therapeutic agents, starting from day 0. Combined ADWA11 (10 mg/kg) and anti-PD1 (10 mg/kg) were injected on days 1, 5, and 9. Immunostaining according to the methods described herein showed effective CD8 clearance (FIG. 21A), which completely reversed beneficial effects (eg, survival and tumor regression) of combination therapy with ADWA11 and anti-PD1 (FIGS. 21B-21C).

These data suggest that CD8+ T cells are important for antitumor effects mediated by anti-avb8, such as ADWA11.

pSmad3, marker of active TGF β signaling is present in CCK168 tumor cells and tumor microenvironment cells, and signaling is significantly inhibited by ADWA11 treatment

Because the best characterized in vivo function of αvβ8 integrin is local activation of latent TGFβ, we decided to determine which cells in untreated tumors exhibit evidence of TGFβ signaling and whether this signaling would be inhibited or suppressed by ADWA11 therapy. To identify cell types in CCK168 tumors that actively signal TGFβ receptors, immunostaining is used with an antibody to phosphorylated SMAD3, a proximal step in TGFβ signaling, as evidence that the cells (CD8+ T cells, F4/80 macrophages, and CD11c dendritic cells) respond to active TGFβ. The CCK168 tumors studied have an extensive network of macrophages (FIG. 22A) in all tumors and only a small number of intercalated DCs. pSMAD3 is readily detected in tumors, usually in cells adjacent to F4/80+ macrophages, but is not detected in macrophages themselves. CD8+ T cells were scattered in untreated mice and were substantially more abundant in tumors from mice treated with AWDA-11. pSMAD3 staining is not observed in these cells. In untreated tumors, high levels of pSMAD3 were visible throughout the tumor microenvironment; however, pSMAD3 staining was broadly inhibited by ADWA11 treatment (FIG. 22A). Thus, without wishing to be bound by any particular theory, these data indicate that in some embodiments, αvβ8 activity is essential for TGFβ activation in these tumors, but that the effect of TGFβ activated by αvβ8 on CD8+ T cell accumulation, Granzyme expression B and the distribution of macrophage subsets is indirect or occurs outside the tumor microenvironment.

Effectorless ADWA11 inhibits tumor growth, improves overall survival, and induces long-term antitumor immunity in the CCK168 squamous cell carcinoma model and CT-26 carcinomas

Initial studies are performed using a native mouse antibody that can interact with Fc receptors. It is therefore possible that the antitumor actions of ADWA11 may arise through antibody-dependent cellular cytotoxicity (ADCC) of tumor cells or tumor-infiltrating macrophages or dendritic cells. To determine the effect of ADCC on ADWA11 activity, a recombinant, Fc "effectorless" version of ADWA11, termed ADWA11_4mut, was generated by introducing 4 substitutions into the mouse IgG1 Fc domain to abolish effector function. These substitutions have previously been shown to completely abolish antibody binding to Fc receptors (Alegre et al ., J. Immunol. 148: 3461-3468, 1992; Hezareh et al., J. Virol. 75: 12161-12168, 2001). ADWA11_4mut is as effective as the wild-type antibody in the CCK168 model (FIG. 28). In addition, the ADWA11_4mut antibody is tested for efficacy in two syngeneic tumor models (ie, CT26 and EMT6 tumor models).

CT-26 cells are selected in addition to CCK168, since the former completely lack detectable expression of αvβ8 (FIG. 18B). CT-26 mouse colon carcinoma cells (ATCC® CRL-2638™) were injected at a dose of 4 x 10 ⁵ cells/mouse subcutaneously into the subcutaneous flank of female Balb/c mice (Charles River Labs). Tumors are grown to a size of 50-100 mm ³ for inclusion in the study. For these studies, ADWA11_4mut or isotype control 2B8_mIgG_4mut is administered by injection on days 0, 4, 8, 12, anti-PD1 antibody (RMP1-14, BioXcell) or isotype control 2A3_rat IgG (BioXcell) is administered by injection intravenously on days 0, 4, 8 All antibodies are dosed in an amount of 10 mg/kg. On day 5, the tumors of all mice, with the exception of the control treatment group without radiation, were targeted with a 5 Gy dose of radiation. Tumor growth is measured twice weekly with digital calipers and reported as volume (length x width x width x 0.5). For the challenge experiment, on day 51 (after the first antibody treatment), complete responder and naïve mice were implanted contralaterally with 2.5 x 10 ⁵ CT26 cells in PRGF and tumor growth was monitored as described above.

In both models, ADWA11_mut4 is effective in driving the antitumor response (FIG. 28), demonstrating that ADCC function is not required for ADWA11-mediated antitumor response.

ADWA11 is effective in a variety of carcinoma models and improves the effects of radiation therapy and anti-PD-1, anti-CTLA-4 and 4-1BB therapies

Determine the effectiveness of ADWA11 in other solid tumor models and whether ADWA11 can more broadly improve the beneficial effects of additional immunomodulatory therapies. The ability of ADWA11 to improve the effect of radiation therapy on CT-26 carcinoma is also being investigated, as this tumor has previously been shown to be radiosensitive, does not express αvβ8 either in vitro or in vivo, and has been shown to respond to a small molecule inhibitor of the TGFB receptor (Young et al., PloS One 11:e0157164, 2016). At doses of radiation that are minimally effective as therapy, addition of either ADWA11_4mut or anti-PD-1 significantly improved tumor regression and overall survival of mice, with 5/9 and 3/10 patients achieving complete therapeutic benefit, respectively (Figure 29A and 29B). Interestingly, the addition of anti-PD1 to ADWA11 adds little additional benefit in this model, providing further evidence that αvβ8 inhibition may be effective even in the absence of checkpoint inhibitors. Surviving mice that showed complete regression of primary tumors and that received either monotherapy or combination therapy were reinfected on the contralateral side with the same CT-26 tumor cells at least 51 days after initial therapy. Minimal tumor growth was observed for contralateral tumors in several mice. All small tumors that initially grew subsequently showed complete regression, indicating that in some embodiments, successful treatment with ADWA11 can induce long-lasting antitumor immunity, as previously described for other immunomodulators (Ascierto et al., J. Transl. Med. 15 :205, 2017) (FIG. 29C). None of the primary tumors that regressed showed regrowth.

ADWA11_4mut is effective in the EMT6 model of breast carcinoma and improves anti-CTLA-4 and anti-4-1BB.

The EMT-6 model of breast carcinoma with immune-excluded tumor microenvironment and low levels of αvβ8 expression is used to study the effect of anti-PD-1, anti-CTLA-4 (which has recently been shown to work through a different molecular mechanism than anti-PD1 ( Wei et al., Cell 170:1120–1133, 2017) or an agonist of 4-1BB, a co-stimulatory receptor expressed on CD8+ T cells (Kang et al., Cancer Res. 2017) in combination with ADWA11_4mut.

ADWA11_4mut is also being tested in the EMT-6 tumor model. 1 X 10 ⁶ EMT6 cells (mouse mammary epithelial carcinoma cell line; ATCC®, CRL2755™) were injected into the quarter of the mammary fat pad of female Balb/c mice (Charles River Labs). Tumors are grown up to 50-100 mm ³ in size. Mice are randomly assigned to antibody treatment groups and operators are not told which treatment group is which. ADWA11_4mut 10mg/kg or control 2B8_mIgG4mut 10mg/kg, anti-CTLA4 (9D9 BioXcell) or isotype control E.tenella-mIgG2b 10mg/kg administered on days 0, 4 and 8 and anti-4-1BB (MAB9371, R&D systems) 1mg /kg is administered on days 0 and 4 intravenously. Tumor growth is measured twice weekly with digital calipers and shown as volume (length x width x width x 0.5).

Anti-PD1, anti-CTLA4 or anti-41-BB monotherapy showed significant initial tumor regression, however, only one mouse treated with anti-4-1BB and one treated with anti-PD1 had complete regression (FIG. 30A-D). In contrast, approximately 70% of mice treated with ADWA11 in combination with anti-PD-1, anti-CTLA4, or anti-4-1BB had complete regression, long-term survival, and resistance to re-challenge with EMT6 tumor cells, suggesting long-term antitumor immunity (FIG .30E). For the challenge experiment, on day 51 (after the first antibody treatment), complete responder and naïve mice were implanted in the contralateral fat pad with 1 X 10 ⁶ EMT-6 cells, and tumor growth was monitored as described above.

These data demonstrate for the first time that ADWA11 antibodies, including ADWA11 2.4, provide a synergistic therapeutic effect when combined with a PD-1 or CTLA4 inhibitor (e.g., an antagonist antibody that binds to PD-1 or CTLA-4 and thereby inhibits the action of PD-1 or CTLA-4, respectively) and/or a 4-1BB agonist (eg, an antibody agonist that enhances the biological activity of 4-1BB). These data suggest that ADWA11 2.4 is an effective human therapeutic agent that can provide a therapeutic antitumor response when combined with an inhibitor, such as PD-1 or CTLA4, or an agonist, such as 4-1BB.

Integrin gene expression α v β 8 and staining in human tumors

The Cancer Genome Atlas (TCGA) study of ITGB8 mRNA expression found that nearly all human tumors of the thirty tumor types examined expressed robust levels of ITGB8 mRNA, with the highest levels of expression found in ovarian carcinoma and renal cell carcinoma (FIG. 23A) . ITGB8 expression in whole tumor lysates may reflect αvβ8 expression on infiltrating immune cells. To test this, multi-panel flow cytometry was performed to assess single-cell αvβ8 protein expression in freshly collected and dissected human tumor sections and biopsies. Analysis of two human ovarian carcinomas and two renal cell carcinomas showed significant αvβ8 expression on CD16+ monocytes, CD14+ tumor-associated macrophages, and monocyte-derived BCDA1+ and BCDA3+ dendritic cells (FIG. 23B).

Discussion

As will be appreciated by those skilled in the art, ADWA11 is a monoclonal antibody specific for blocking the αvβ8 integrin and is an effective antitumor immunotherapy when used alone. For example, ADWA11 demonstrated antitumor activity in CCK168 cutaneous squamous cell carcinoma. Additionally or alternatively, ADWA11 in combination with an immunomodulator (eg, anti-PD-1, anti-CTLA-4, and anti-4-1BB) or radiation therapy demonstrates effective and synergistic enhanced antitumor activity in three syngeneic allograft models of epithelial carcinomas in three different mouse strains. In some embodiments, inhibition of αvβ8 increases the number of CD8+ T cells in tumors, their cytotoxic differentiation, as measured by Granzyme B expression in these cells. Elimination of CD8+ T cells abolishes the antitumor effects of ADWA11 and anti-PD1 treatment, indicating that in some embodiments, improved tumor cell killing by CD8 T cells is critical to the effectiveness of ADWA11. In addition to its effects on CD8+ T cells, ADWA11 increases the number of immunostimulatory monocytes in the tumor microenvironment (Franklin et al., Science 344:921-925, 2014; Movahedi et al., Cancer Res . 70:5728-5739, 2010; Ostuni, Trends Immunol 36: 229-239, 2015; Noy et al., Immunity 41:49-61, 2014). Although there was no difference in the number of immunosuppressive macrophages, these macrophages, along with dendritic cells, expressed high levels of detectable αvβ8. Although the first tumor line tested, CCK168 cells, expressed significant surface levels of αvβ8 in vitro , little αvβ8 expression was detected by flow cytometry on non-hematopoietic cells from harvested tumors, indicating that in some embodiments expression is lost when these malignant cells form tumors in vivo . However, the possibility that the antitumor effects of ADWA11 were due to targeting the malignant cell directly through ADCC was ruled out by replicating the results using a recombinant, effectorless ADWA11 antibody in cells that do not express any detectable αvβ8 (i.e., colon carcinoma cells CT-26). The effectorless antibody was able to suppress tumor growth in three tumor models, CCK168, CT-26, and EMT-6, of which CT-26 had undetectable αvβ8 expression. In addition, short-term ADWA11 therapy resulted in long-lasting tumor suppression and resistance to subsequent re-challenge, indicating, in some embodiments, the induction of long-lasting antitumor immunity. Together, these data indicate that, in some embodiments, blockade of αvβ8 integrin causes tumor suppression by blocking αvβ8 on innate immune cells or regulatory T cells to enhance adaptive immunity to tumors.

Activation of TGFβ through αvβ8 expressed on dendritic cells, regulatory T cells, fibroblasts, airway epithelial cells and neuroepithelium has been shown (Travis et al., Nature 449:361-365, 2007; Melton et al., J. Clin. Invest 120:4436-4444, 2010; Arnold et al., J. Neurosci. 32(4):1197-1206, 2012; Fenton et al., Mucosal Immunol. 10:624-634, 2017; Mu et al., J. Cell Biol. 157:493-507, 2002; Proctor et al., J. Neurosci. 25:9940-9948, 2005; Edwards et al., J. Immunol. 193:2843-2849, 2014), but the definition the full range of αvβ8 integrin expression has been limited by the lack of reliable tissue staining reagents. In the examples provided herein, newly developed antibodies capable of detecting αvβ8 integrin by flow cytometry were used to demonstrate that tumor-associated macrophages and dendritic cells are the main cell types exhibiting high cell surface staining for αvβ8 in mouse carcinomas. This indicates that, in some embodiments, expression on one or more of these cell types is important for αvβ8-mediated suppression of local antitumor immunity.

In the Examples provided herein, expression of αvβ8 on T cells, including regulatory T cells (Tregs), was not detected.

Activation of TGFβ by integrins, including αvβ8, is tightly spatially restricted, allowing cells expressing αvβ8 to present TGFβ locally to cells with which they contact directly (Travis et al., Nature 449:361-365, 2007; Munger et al., Cell 96: 319-328, 1999). TGFβ has been described to suppress the activity of effector T cells. As shown in the examples herein, CD8+ T cells are increased in ADWA11-treated tumors, are more likely to express Granzyme B, and are important in mediating the protective effects of ADWA11. In some embodiments, the suppressive effect of αvβ8 on innate immune cells is due to direct presentation of active TGFβ to CD8 T cells. However, immunostaining for pSMAD3 showed no evidence of TGFβ signaling in CD8+ T cells, but showed robust signaling in non-hematopoietic cells (eg, tumor cells). Thus, in some embodiments, tumor cells and certain other tumor-associated non-hematopoietic cells (eg, fibroblasts) are functionally important cells that respond to TGFβ activated by αvβ8 and suppress local tumor immunity.

As described in the examples herein, αvβ8 integrin is widely expressed on monocytes, macrophages and dendritic cells in a variety of mouse and human tumors and is an effective modulator of the antitumor immune response. Monotherapies (eg, ADWA11) targeting this integrin are effective in some tumors. Additionally, efficacy is synergistically enhanced by combining αvβ8 inhibition (eg, ADWA11 treatment) with either a checkpoint inhibitor (eg, anti-PD1 or anti-CTLA4) or an immune activator (eg, anti-4-1BB) or by combining αvβ8 monotherapy with radiation therapy. Taken together, these results identify αvβ8 integrin as a novel target for tumor immunotherapy.

Example 15: Additional in vivo ADWA11 rating 2.4

Brief description of tumor models for anti- α v β 8 ratings

In vivo efficacy is assessed using syngeneic tumor models in immunocompetent mice. Efficacy studies are being conducted with an effectorless version of the parent mouse hybridoma antibody ADWA11_4mut due to the strong anti-species drug response that limited the exposure of humanized ADWA11 2.4.

Table 9. Tumor models for anti- α v β 8 assessment

Model Tumor microenvironment Expression of αV integrins TGFβ pathway status EMT6, breast cancer Model of tumor microenvironment with immune exclusion Expresses both αvβ8 and αvβ6 High TGFβ pathway activation gene expression profile CT26, colon cancer Pre-existing immune model Does not express αvβ8 or αvβ6 Low TGFβ pathway activation gene expression profile

Efficiency in the EMT6 model

The EMT6 tumor model study is performed orthotropically (4th breast fat pad) and anti-αvβ8 treatment with or without checkpoint inhibitors is performed simultaneously.

Charles River Balb/c mice (n=10 per group) were implanted with 3x105 or 1x106 EMT6 cells/mouse. Treatment begins with an average tumor volume of 50 mm ³ or 100 mm ³ .

The parent anti-αvβ8 mouse antibody ADWA11_4mut was dosed at 10 mg/kg once every fourth day for a total of three doses in all studies. Anti-PD1 antibody (clone RMP1-14, BioXcell) dosed at 10 mg/kg once every four days for a total of three doses, 4-1BB agonist mAb (clone MAB9371, R&D systems) dosed at 1 mg/kg one once every four days for a total of two doses and anti-CTLA4 (clone 9D9, BioXcell) is dosed at 10 mg/kg once every four days for a total of three doses.

Anti-αvβ8 monotherapy efficacy of ~50% tumor growth inhibition (TGI) (days 10-20) is observed when 3x10 ⁵ cells are implanted and treatment is started at 50 mm ³ . However, when 1x10 ⁶ cells are implanted and treatment is started at 100 mm ³ , no RRO for anti-αvβ8 monotherapy is observed. The difference in TRI for anti-αvβ8 monotherapy between the two studies was unclear; however, without wishing to be bound by any particular theory, the rapid rate of tumor growth in this model when implanted with 1x10 ⁶ cells may limit the response or outpace the antitumor response to αvβ8 blockade. Importantly, anti-αvβ8 demonstrates significant synergistic therapeutic effects with anti-PD1 (7/10 complete response), 4-1BB agonist (5 complete responses) and anti-CTLA4 (6 complete responses) and provides increased survival compared with monotherapy and isotype control. Therefore, these data suggest that ADWA11 antibodies may mediate an antitumor response in the EMT-6 tumor model.

Anti-CTLA4 or 4-1BB agonist monotherapy demonstrates significant responses (~50% RTI, days 8–20); however, the response is temporary and tumors eventually outgrow the response. To demonstrate that the combination treatment induces a tumor EMT6 cellular response, compared with an anti-angiogenic or anti-proliferative response, mice with a complete response were rechallenged on day 51 (after the 1st dose) with a second EMT6 tumor implant, but without additional drug treatment. EMT6 tumors grow rapidly in naïve mice but clear rapidly in complete response mice, indicating, in some embodiments, the development of cellular immunity to tumor cells, such as direct tumor cell death not mediated by anti-angiogenesis or anti-proliferation.

Quantification of lymphocyte abundance in the EMT6 model tumors

To investigate the effect of anti-αvβ8 treatment on the EMT6 tumor microenvironment, the abundance of lymphocyte subsets was quantified using IHC. Briefly, Charles River Balb/c mice (n=10 per group) were implanted with 1x10 ⁶ EMT6 cells and treatment was initiated at an average tumor volume of 100 mm ³ (Day 0). Anti-αvβ8 ADWA11 2.4 antibody is dosed at 10 mg/kg on every third day (eg Day 0, Day 3, Day 6 and Day 9) for a total of four doses, and tumors are harvested on Day 11 11 (48 hours after 4 th dose).

Immunohistochemical (IHC) analysis of staining densities for CD45 (total lymphocytes and myeloid cells), CD3 (total T cells), CD4 T cells, CD8 T cells, and Granzyme B (activated CD8 and NK cells) demonstrates that anti-αvβ8 monotherapy increases the number of total CD45, CD4 T cells, CD8 T cells and very significantly reduced the density of cells expressing Granzyme B (n=10 for each group) (FIG. 15). IHC data indicate that in some embodiments, ADWA11 2.4 monotherapy is sufficient to alter the tumor microenvironment, consistent with the expected mechanism of action (OMA) of αvβ8.

Efficiency in the CT26

The CT26 model was selected based on lack of αvβ8 expression, partial response to anti-PD1 therapy, and literature evidence of a synergistic RRI for a small molecule TGFβ inhibitor with a suboptimal radiation dose. Radiation therapy (RT) is of particular interest due to its ability to induce immunological cell death and de novo priming of DC-T cells, where αvβ8 potentially plays an important role in shaping T cell differentiation and activation. Briefly, Charles River Balb/c mice (n=10 for each group) were implanted subcutaneously with 4e5 cells and treatment was initiated at an average tumor volume of 100 mm ³ .

Anti-αvβ8 effector null mouse antibody ADWA11_4mut is administered at a dose of 10 μg/kg Q4Dx4, anti-PD1 (clone RMP1-14, BioXcell) is dosed at 10 μg/kg Q4Dx3 and a dose of 5 Gy of tumor-targeted radiation is given 5 days after first dose of mAb. Incorporation of anti-αvβ8 with RT resulted in a significant increase in IRO, with 5/9 mice showing a complete response. Incorporation of anti-PD1 with RT resulted in less significant RRI compared to anti-αvβ8, with 3/10 mice showing a complete response. The triple combination of RT, anti-αvβ8 and anti-PD1 produced a very significant IRO in 7/10 mice with a complete response. As in the EMT6 study, mice with a complete response were immune to rechallenge with CT26 cells.

Immunophenotyping of the cell population infiltrating the CT26 tumor

To investigate the effect of anti-αvβ8 treatment on the CT26 tumor microenvironment, the abundance of lymphocyte subsets was quantified by flow cytometry. Briefly, Charles River Balb/c mice (n=6 for each group) were implanted subcutaneously with 4e5 cells and treatment was initiated at an average tumor volume of 100 mm ³ . Anti-αvβ8 ADWA11 2.4 is dosed at 10 mg/kg or 1 mg/kg Q3Dx3, and tumors are harvested on day 8 (48 hours after the 3rd dose). Tumors are dissociated and the lymphocyte population is quantified by CyTOF cytometry. Anti-αvβ8 (10 and 1 mg/kg) increases the number of CD8 T lymphocytes in the CD3 T cell gate. In addition, CD8 cells more frequently express GranzymeB, a marker of the activated phenotype.

Essence

Taken together, the efficacy and PD studies of EMT6 and CT26 demonstrate: (1) that anti-αvβ8 synergistically increases the response to multiple checkpoint inhibitors; (2) that the effectiveness of anti-αvβ8 is independent of tumor cell expression of αvβ8; and (3) that anti-αvβ8 ADWA11 2.4 monotherapy is sufficient to increase the abundance of CD8+ GzmB+ T cells in the tumor microenvironment.

Example 16: Pharmacokinetics of ADWA11 2.4 in Human FcRn Transgenic (TG32) Mice

The TG32 mouse model has been shown to be as good as monkey models for predicting the pharmacokinetics of IgG1 and IgG2 mAbs in humans, which exhibit linear pharmacokinetics. The pharmacokinetics of ADWA11 2.4 were assessed in TG32 mice following a single IV bolus dose administered at 0.1, 0.3 or 3 mg/kg. The drug concentration in the serum is then measured. As shown in FIG. 25A and FIG. 25B, a clear trend toward a dose-dependent decrease in antibody clearance is observed. This is typical for monoclonal antibodies that interact with cell surface targets and suggests that the target receptor, αvβ8, may act as a clearance mechanism. Therefore, two separate pharmacokinetic models are used to describe the linear (typical FcRn-mediated antibody clearance mechanism) versus non-linear (αvβ8-mediated) clearance pathway. To estimate linear clearance parameters, a two-compartment population pharmacokinetic model was used to fit only the 3 mg/kg data (FIG. 25A). To estimate non-linear clearance pharmacokinetic parameters for multiple doses, a two-chamber saturated model (Michaelis Menten) was used to fit all data (0.1, 0.3, 3 mg/kg) (FIG. 25B). The calculated linear and non-linear pharmacokinetic parameters are shown in Tables 10 and 11, respectively. Linear pharmacokinetic parameters are within the range observed for typical mAbs in TG32 mice.

Table 10. Dual-compartment pharmacokinetic parameters in TG32 mice (IV at a dose of 3 mg/kg)

Parameter Grade %CV CL (ml/h/kg) 0.33 9.2 CLD (ml/h/kg) 2.3 13 V1 (ml/kg) 49 2.4 V2 (ml/kg) 38 9.6 t _1/2 (day) 7.6

CL: clearance from the central chamber; CLD: interchamber clearance distribution; V1: distribution volume for the central chamber; V2: volume of distribution for the peripheral chamber; t _1/2 , terminal half-life calculated from estimated parameters.

Table 11. Two-chamber non-linear pharmacokinetic parameters in TG32 mice (IV at a dose of 0.1, 0.3 and 3 mg/kg)

Parameter Grade %CV Km (ng/ml) 1659 25 Vmax (ng/h/kg) 6596 5 CLD (ml/h/kg) 3.4 20 V1 (ml/kg) 49 4 V2 (ml/kg) 48 8

Vmax: maximum speed; Km: substrate concentration to achieve half-maximum speed; CLD: interchamber clearance distribution; V1: distribution volume for the central chamber; V2: volume of distribution for the peripheral chamber.

Consistent with observed target-mediated drug distribution (TMDD) based on serum pharmacokinetics, a dose-dependent decrease in drug distribution to multiple tissues (due to saturable target binding), especially the kidney, is also observed in the tissue distribution study.

Example 17: Pharmacokinetics in Non-Human Primates (NHP)

The pharmacokinetics of ADWA11 2.4 were evaluated in an NHP diagnostic toxicology study after the first dose in a multiple-dose study at 4, 40, or 100 mg/kg. As shown in FIG. 26, the effects of ADWA11 2.4 appear to be linear within this dose range due to it being above the saturation range for target-mediated drug distribution (TMDD). A two-compartment linear model was used to fit the data (FIG. 26), and the estimated pharmacokinetic parameters are shown in Table 12. While the observed CL volumes and distributions were within the ranges for typical mAbs, the final t _1/2 appears to be at the short end of the range. Without wishing to be bound by any particular theory, this is likely due to the fact that the end-point phase was not clearly defined, due to sampling only up to 7 days post-dose (the limit of the ETS study design).

Table 12. Two-compartment pharmacokinetic parameters in NHP (IV at a dose of 4, 40 and 100 mg/kg - first dose)

Parameter Grade %CV CL (ml/h/kg) 0.41 8.7 CLD (ml/h/kg) 15.9 25 V1 (ml/kg) 35.6 12 V2 (ml/kg) 25.1 25 t _1/2 (day) 4.3

CL: clearance from the central chamber; CLD: interchamber clearance distribution; V1: distribution volume for the central chamber; V2: volume of distribution for the peripheral chamber; t _1/2 , terminal half-life calculated from estimated parameters.

Example 18: Pharmacokinetics of Murine Surrogate Antibody as Monotherapy and Co-Dosed with Anti-PD-1

Pharmacokinetic data for a single dose of the parent (mouse IgG) are not available, but effects have been measured up to four time points in several efficacy studies and appear to be consistent across these studies. The extent of exposure of the parent (mouse IgG) appears to be linear over the dose range tested (1, 3 and 10 mg/kg), consistent with TMDD saturation as assessed in TG32 mice for the humanized antibody, ADWA11 2.4. The two-compartment pharmacokinetic model is compared to the effect rate data in a dose-response study of the EMT6 tumor model, assessing only the CL while fixing the other parameters to the values shown above for the humanized antibody in TG32 mice. The estimated baseline (mouse IgG) CL in these tumor-bearing mice is 0.74 ml/h/kg.

The extent of exposure to the parent (mouse IgG) appears to be ~5-50x lower in a repeated co-dosing study with anti-PD-1 antibody compared to that of dosing with the surrogate mouse antibody alone or co-dosing with control rat IgG 2A3. This phenomenon was observed in two separate studies using two different tumor models (CT26 and EMT6). The lower effect is likely due to increased ADA formation in the presence of anti-PD-1, as has been shown in PD-1 knockout mice (Nishimura H et al., 1998, International Immunol. 10(10): 1563-72). Increased ADA formation following treatment with anti-PD-1 antibody (pembrolizumab) has also been shown in the clinic.

Example 19: Prediction of PK/Exposure in Humans

The pharmacokinetic profile of ADW11 2.4 in humans at doses saturating target-mediated drug distribution (TMDD) is predicted by scaling linear pharmacokinetic parameters from TG32 mice (Table 13). The predicted linear human pharmacokinetic parameters are given in Table 13. Additionally, a human linear CL of 0.12 ml/h/kg is predicted based on measured AC-SINS binding (score=2, section 2.4), using the PBPK model platform. A human linear CL of 0.086 mL/h/kg is predicted by allometric scaling from NHP. These CL values are within the 2x range of prediction based on TG32 allometric scaling (0.15 mL/h/kg), further confirming that at TMDD-saturating doses, ADW11 2.4 will have a pharmacokinetic profile that is typical of a monoclonal antibody.

These results indicate that ADWA11 2.4 is a potential useful therapeutic antibody for humans.

Table 13. Predicted linear human pharmacokinetic parameters for ADWA11 2.4

Parameter Scaling factor Predicted value CL (ml/h/kg) 0.9 0.15 CLD (ml/h/kg) 0.67 0.15 V1 (ml/kg) 0.97 39 V2 (ml/kg) 0.93 21 t _1/2 (day) ND 12

CL: clearance from the central chamber; CLD: interchamber clearance distribution; V1: distribution volume for the central chamber; V2: volume of distribution for the peripheral chamber; t _1/2 , terminal half-life calculated from estimated parameters.

Additional PK and/or TK for a single IV and PC dose of ADWA11 (2.4) were characterized in male hFcRn TG32 mice (n=4 or 8/dose group) and male cynomolgus monkeys (n=1/dose group) and adjusted for human pharmacokinetic parameters mentioned earlier in this document. More specifically, following single IV dosing (0.1, 0.3, and 3 mg/kg TG32 in mice and cynomolgus monkeys), the PK of ADWA11 (2.4) was not linear in both species, with a trend toward a dose-dependent decrease in CL, consistent with target saturability. indirect distribution of the drug. At doses above saturable clearance, the PK of ADWA11 (2.4) is linear and consistent with typical human IgG1 mAb. Mean PK and TK parameters following a single PC dose of 10 mg/kg, T _max observed 240 hours after dosing, and bioavailability estimated to be approximately 100% based on comparison with dose-normalized AUC _inf following a single IV dose of 3 mg/kg kg.

ADWA11 (2.4) is expected to be dose dependent at lower doses than stated above elsewhere in this document. That is, the human PK of ADWA11 (2.4) is predicted based on allometric scaling of the cynomolgus monkey PK model, which includes both linear and non-linear clearance. Adjusted predicted PK parameters for humans are as follows: 36 ml/kg for central chamber volume of distribution, 33 ml/kg for peripheral chamber volume of distribution, 0.12 ml/kg/h for central chamber clearance (linear CL), 0. 51 ml/kg/h for intercompartmental clearance and a maximum non-linear elimination rate of 0.46 µg/ml/h, and Km 0.42 µg/ml for non-linear clearance. This model predicts that the non-linear clearance of ADWA11 (2.4) is likely to be saturated at plasma concentrations above 14 µg/mL with a predicted t½ of approximately 15-17 days.

Example 20: Pharmacokinetics-pharmacodynamics relationship and prediction of effective human dose

In the absence of clear responses to the extent of IRO exposure in syngeneic mouse tumor models, a dose of 10 mg/kg is considered the effective dose to estimate the effective concentration. Using a pharmacokinetic model for parenteral administration (mouse IgG), the mean concentration (C _avg ) was estimated to be 107 μg/ml on day 12 (4 days after the last dose in the Q4D study x 3 doses of 10 mg/kg). Also, although slightly higher, C _avg values are estimated by calculating the AUC using the linear trapezoidal rule from available exposure data.

Because typical antibody pharmacokinetics are expected for ADWA11 2.4 at these effective concentration levels, a two-compartment pharmacokinetic model with scaled mouse TG32 parameters is used to predict the human dose (Table 13) (Betts et al. MABS (2018) 1-14). In addition, human doses predicted using scaled pharmacokinetic parameters for NHP differ by only about 20% from doses predicted using mouse TG32. To fit C _avg , an effective human IV dose of 7 mg/kg is predicted for the Q14D x 3 dosing, and an IV dose of 12 mg/kg is predicted for the Q28D x 3 dosing (Figures 27A-27B). The dosage may also be tailored to specific patient populations, clinical indications, and/or clinical signs and symptoms.

ADWA11 2.4 human PK prediction was refined based on allometric scaling from the cynomolgus monkey and results from additional preclinical efficacy studies. The predicted effective dose (C _eff ) is defined as the plasma concentration that is 10 times the half-maximal effect on tumor growth inhibition in various studies using the above 2 mouse tumor models. C _eff in all studies was in the range of 10–98 nM; therefore, a conservative approach was used to define 100 nM (upper range limit) as the target _Ceff . The effective dose of ADWA11 2.4 is predicted to be approximately 2 mg/kg Q14D IV or 4 mg/kg Q28D EV, which is predicted to provide >IC90 tumor growth inhibition coverage at the estimated trough drug concentration (100 nM). Predicted _Cmax , C _ave and _Cmin at steady state at predicted effective doses are 84, 44 and 29 µg/ml after 2 mg/kg Q14D (BB) and 132, 45 and 21 µg/ml after 4 mg/kg Q28D ( BB), respectively. The predicted t½ for humans is approximately 15-17 days at the predicted effective dose.

These results confirm that ADWA11 2.4 is a potentially useful therapeutic antibody for humans in that it can be dosed in quantities that are economically feasible and suitable for production.

Example 21: Repeated Administration Toxicokinetics in CD-1 Mice

Toxicokinetics and drug antibody assessments are performed following weekly intravenous (IV) or subcutaneous (SC) administration of ADWA11 2.4 at 10 (IV), 100 (SC) or 200 (SC) mg/kg/week for a total of 4 doses in CD mice -1 (n=3/sex/dose per group) as part of a GLP repeat dose toxicity study.

There were no quantifiable concentrations of ADWA11 2.4 in samples collected and analyzed from the vehicle controls at any time point during the study. Based on a qualitative review of the data, there were no consistent sex differences in systemic exposure (as assessed by Cmax and AUC168), so group average toxicokinetic parameters are presented using pooled data from male and female CD-1 mice (Table 18).

Table 18. Overall mean toxicokinetic parameters for ADWA11 2.4 in CD-1 mice.

Dose (mg/kg/week)/route Day C _max
(µg/ml) _Tmax
(watch) AUC ₁₆₈ (µg•h/ml) 10/BB 1
22 199
615 0.54
1.1 15400
46400 100/pcs 1
22 871
750 72
18 104000
591000 200/BB 1
22 5020
6510 0.25
0.54 258000
267000

AUC ₁₆₈ = Area under the concentration-time curve from 0 to 168 hours; C _max =maximum observed concentration; T _max = time at which C _max is first observed.

Following IV dosing, systemic exposure increases with increasing dose in an approximately dose-dependent manner on day 1, and in a less-than-dose-dependent manner on day 22. Mean accumulation rates (AUC ₁₆₈ , Day 22/Day 1) were 3.0, 0.6 and 1.0 for 10 mg/kg/week (BB), 100 mg/kg/week (WK) and 200 mg/kg /week (WW), respectively. The lack of accumulation after repeated dosing at 100 mg/kg/week (SC) and 200 mg/kg/week (WS) may be due to the presence of ADA in animals in these dosing groups.

The overall frequency of ADA induction to ADWA11 2.4 was 28% (5/18 animals). The frequency of ADA induction to ADWA11 2.4 was 0% (0/6 animals), 67% (4/6 animals), and 17% (1/6 animals) in CD-1 mice dosed with ADWA11 2.4 10 (IV), 100 (PC) or 200 (BB) mg/kg/week, respectively. Overall, exposure in ADA-positive animals was the same or lower than for ADA-negative animals.

Example 22: Repeated administration toxicokinetics in cynomolgus monkeys

Toxicokinetics and drug antibody assessments are performed following weekly intravenous (IV) or subcutaneous (SC) administration of ADWA11 2.4 at doses of 8 (IV), 100 (SC) and 200 (SC) mg/kg/week for a total of 5 doses in cynomolgus monkeys as part of a repeated dose toxicity study of GLP.

There were no quantifiable concentrations of ADWA11 2.4 in samples collected and analyzed in the vehicle control group at any time point during the study or in any group dosed with study drug prior to dosing on day 1. There were no obvious sex differences in systemic exposures. (estimated by C _max and AUC ₁₆₈ ) by dosage group; therefore, group mean TK parameters are discussed and presented using pooled data for male and female cynomolgus monkeys (Table 19).

Table 19. Overall mean toxicokinetic parameters for ADWA11 2.4 in cynomolgus monkeys .

Dose (mg/kg/week)/route Day C _max
(µg/ml) _Tmax
(watch) AUC ₁₆₈ (µg•h/ml) 8/BB 1
22 248
394 0.54
6.3 17300
44600 100/pcs 1
22 1270
2880 72
32 161000
393000 200/BB 1
22 6200
11400 1.1
2.7 479000
1100000

AUC ₁₆₈ = Area under the concentration-time curve from 0 to 168 hours; C _max =maximum observed concentration; T _max = time at which C _max is first observed.

Following weekly dosing at 8 (IV), 100 (SC), and 200 (IV) mg/kg, systemic exposure increases approximately proportional to dose on both Day 1 and Day 22, with accumulation factors (Study Day 22/Study Day 1 ) ranging from about 2.3 to 2.6.

The overall rate of ADA induction to ADWA11 2.4 was 39% (7/18 animals) for all dosing groups. The rates of ADA induction to ADWA11 2.4 were 17% (1/6), 33% (2/6), and 67% (4/6) in animals dosed with ADWA11 2.4 8 (IV), 100 (SC), or 200 ( IV) mg/kg/week, respectively. Overall, the effects on ADA-positive animals were similar compared to ADA-negative animals.

Example 23: Non-clinical toxicology in mice and cynomolgus monkeys

AWA11 2.4 was administered to mice and cynomolgus monkeys in intravenous (IV) and subcutaneous (SC) studies for up to 1 month. Target organs identified in these studies include bone, spleen, and clinical chemistry changes, although these changes are not considered adverse. The dose not leading to the development of an observed adverse effect (NOAEL) in 1-month studies was 200 mg/kg/week IV (C _max and AUC ₁₆₈ 6510 μg/ml and 267000 μg⋅h/ml) for mice and (C _max and AUC ₁₆₈ 11,400 µg/ml and 1,100,000 µg⋅h/ml) for cynomolgus monkeys, respectively.

Single dose toxicity

ADWA11 2.4 is tolerated following a single PT dose of 10, 30, or 100 mg/kg in female CD-1 mice after a 2-week observation phase. No data related to the test drug were available during the study, and mean systemic exposure increased with each dose in a dose-dependent manner, as measured by area under the curve (AUC). At 100 mg/kg, the average _Cmax and AUC ₃₃₆ were 795 μg/ml and 148,000 μg*h/ml, respectively.

Repeated dosing toxicity

Study 1 : In Study 1, female CD-1 mice were administered ADWA11 2.4 IV bolus at doses of 0, 1, 10, or 100 mg/kg/dose on days 1, 4, 8, 11, and 14. All mice survived the study and all doses are transferred. At 100 mg/kg/dose, there are test substance-related differences in clinical chemistry parameters compared to control means, which include decreased glucose (0.75x) and increased phosphorus (1.35x), globulin (1.09x) and nitrogen blood urea (1.18x) without any associated microscopic data or clear relationship to dose (glucose and phosphorus). At ≤10 mg/kg/dose, test substance-related differences in clinical chemistry parameters included low glucose (0.77x at 10 mg/kg/dose only) and high phosphorus (1.26x), compared with control means. Mean systemic exposure increased with increasing dose in an approximately dose-dependent manner on days 1 and 11. At 100 mg/kg/dose, the highest dose administered, mean C_max and AUC48 are 4510 μg/ml and 124,000 μg⋅h/ml on day 11.

Study 2 : In Study 2, cynomolgus monkeys were administered ADWA11 2.4 IV bolus once weekly at doses of 0, 4, 40, or 100 mg/kg/dose on days 1, 8, and 14. All monkeys survived the study and all doses were tolerated. The only test substance-related data were decreases in CD8+ naïve T cells in both female (0.21x baseline) and male (0.44x baseline) monkeys in the 100 mg/kg/dose group, but no changes. observed in the 4 or 40 mg/kg/dose groups. Mean systemic exposure increases with increasing dose in an approximately dose-dependent manner over 1 day. At 100 mg/kg/dose, the highest dose administered, mean C_max and PPK₁₆₈ are 3550 µg/ml and 162,000 µg⋅h/ml on day 1.

Study 3 : In Study 3, ADWA11 2.4 was administered to male cynomolgus monkeys by IV bolus once every 1 day at doses of 0.1, 0.3 or 3 mg/kg/dose or PC injection on 1 day at doses of 0.1, 0.3 or 3 mg/kg or PC injection 10 mg/kg. Animals treated with 0.1 or 0.3 mg/kg (IV) on day 1 were then also given 30 or 100 mg/kg PC by injection, respectively, on day 8. All administered doses and routes of administration were tolerated. There are no lifetime observations associated with the test substance (ie, clinical signs or changes in body weight or food intake) during the 22- or 29-day post-dose observation period. There is no evidence of redness or swelling at the injection sites.

After a single IV dose of 0.1, 0.3, and 3 mg/kg on day 1, systemic drug exposure increased in a dose-dependent manner between 0.1 and 0.3 mg/kg, and more than in a dose-dependent manner between 0.3 and 3 mg. /kg. After a single PC administration of 10 mg/kg on day 1, or at 30 mg/kg and 100 mg/kg on day 8, the systemic exposure of the drug increases in a dose-dependent manner. PC bioavailability relative to an IV dose of 3 mg/kg is 103%, 79% and 100% at 10, 30 and 100 mg/kg, respectively. Administration of ADWA11 2.4 at 0.1, 0.3, or 3 mg/kg (IM) or 10, 30, or 100 mg/kg (SC) to cynomolgus monkeys was tolerated, and systemic exposure increased with dose.

Study 4 : In Study 4, ADWA11 2.4 was administered once weekly to male and female CD-1 mice with IV and/or PC at doses of 0 (IV and SC), 10 (IV), 100 (PC) or 200 (IV) mg/kg /week for 1 month (total 5 doses). Data associated with the tested substance were limited to benign changes in clinical chemistry, spleen immunophenotyping parameters, and spleen weight.

All animals survived until scheduled necropsy, with the exception of one male 100 (PC) mg/kg per week, which was found dead on day 5; however, the death of this animal is not considered to be related to the test substance. Postmortem autolysis is observed in various organs and was the cause of death in the 200 (BB) mg/kg/week group, and included higher globin (1.15x to 1.16x) in both sexes, lower AG ratio (0.85x) in females and higher total protein content (1.11x) in males compared to concurrent controls. Administration of the test mAb increases systemic gammaglobulin concentrations and likely contributes to these changes in serum proteins. Because of their small magnitude and lack of corresponding microscopic or macroscopic findings, these observations in clinical chemistry parameters were not unfavorable. In the spleen, test substance-associated lower numbers of CD8+ T cells (0.52x, 0.65x and 0.60x control mean) in females and a higher percentage of CD4+ T cells expressing the activation marker CD25 (1.62x, 1. 90x and 1.52x control mean) in males were observed at doses of 10 (BB), 100 (PC) and 200 (BB) mg/kg/week, respectively, on day 30. Mean absolute and relative (to body and brain weight) spleen weight was lower (from 0.86x to 0.88x control) in females at a dose of 200 (BB) mg/kg/week. There were no correlating macroscopic or microscopic findings for lower spleen weights. Based on a qualitative review of the data, there were no consistent sex-related differences in systemic exposure as assessed by _Cmax and AUC ₁₆₈ . Systemic exposure increased with increasing IV or PC dose. After repeat dosing, exposures increased at 10 mg/kg/week (IV), decreased at 100 (SC) mg/kg/week, and remained the same at 200 (TV) mg/kg/week. Anti-drug antibody (ADA) induction rates to ADWA11 2.4 were 0% (0/6), 67% (4/6), and 17% (1/6) at 10 (BB), 100 (PC), or 200 (BB) mg /kg/week, respectively. There was no evidence of ADWA11 2.4-related microscopic findings in the heart or skin one month after ADWA11 2.4 administration.

After repeated dosing, exposure in ADA-positive animals was similar or slightly lower compared to ADA-negative animals. The highest dose administered, 200 (IV) mg/kg/week, was defined as the no observed adverse effect dose (NOAEL) based on the absence of adverse effects at any dose and was associated with a _{Cmax of} 6510 μg/mL and an AUC _{of 168} from 267,000 mcg⋅h/ml on day 22.

Study 5 : In Study 5, cynomolgus monkeys were administered ADWA11 2.4 as IV and/or SC doses of 0 (IV and SC), 8 (IV), 200 (IV) or 100 (SC) mg/kg/week for 1 month (total 5 doses). There were no adverse outcomes associated with the test substance in this study. Non-adverse findings associated with the test substance include unilateral physical dysplasia at the costochondral junction in males and changes in serum proteins (increased globulin (up to 1.29x), total protein (up to 1.10x) and/or decreased albumin ratio and globulins (up to 0.75x) in females at 100 (PC) mg/kg/week and both sexes at 200 (BB) mg/kg/week Systemic exposure (estimated C_max and PPK₁₆₈) was similar in males and females across dosing groups, and mean systemic exposure in the IV dose groups increased with increasing dose in an approximately dose-dependent manner on days 1 and 22. Exposures were higher after repeated doses in all groups. There was no evidence of ADWA11 2.4-related microscopic findings in the heart or skin one month after ADWA11 2.4 administration.

ADA induction rates were 17% (1/6 animals), 33% (2/6 animals), and 67% (4/6 animals) for animals receiving ADWA11 2.4 at a dose of 8 (IV), 100 (SC), or 200 ( IV) mg/kg/week, respectively; the rate of ADA induction to ADWA11 2.4 was 39% in all groups. Serum exposure in ADA-positive animals was generally similar compared to ADA-negative animals. Following IV or SC administration once weekly, ADWA11 2.4, 200 (IV) mg/kg/week was determined to be a NOAEL in this 1-month toxicity study in monkeys, based on the absence of adverse findings in any in-living or post-mortem studies. . At 200 (IV) mg/kg/week, the mean C _max is 11,400 μg/mL and the mean AUC ₁₆₈ is 1,100,000 μg⋅h/mL on day 22.

NOAEL in 1-month studies is 200 mg/kg IV for both mice ( _Cmax and AUC _last 6510 µg/ml and 267000 µg⋅h/ml) and cynomolgus macaques ( _Cmax and AUC _last 11400 µg/ml and 1100000 µg ⋅h/ml), respectively.

Example 24: In vitro binding of complement protein C1q and FcR

The potential of ADWA11 2.4 to cause complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) was examined in in vitro screening assays through C1q and Fc gamma receptor (FcγR) binding, respectively. ADWA11 2.4 does not bind to C1q at concentrations tested (up to 30 μg/ml) and is therefore considered to have low potential for CDC induction. Binding of ADWA11 2.4 to all FcγRs tested was similar or lower than binding to the negative control antibody and lower than binding to the positive control antibody. Thus, ADWA11 2.4 is considered to have low potential to induce ADCC activity.

Example 25: In vitro cytokine release assay

In vitro cytokine release assays were performed in whole blood and PBMC formats using samples from 8 human donors. After incubation with ADWA11 2.4, no TNF, IL-6 or INF-γ release associated with the test substance was observed. These data are consistent with the lack of changes in serum cytokine profiles following ADWA11 2.4 administration in the mouse and monkey studies described above.

Example 26: α v β 8 Inhibition Improves Efficacy of Anti-PD-1 Therapy in MC38 Tumor Model

Methods:

In this study, the mouse colon carcinoma cell line MC38 was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 100 units/ml sodium penicillin G, 100 μg/ml streptomycin sulfate and 25 µg/ml gentamicin. Cells are cultured in tissue culture flasks in a humidified incubator at 37°C under an atmosphere of 5% CO ₂ and 95% air. On the day of tumor implantation, MC38 tumor cells are harvested during exponential growth and resuspended in phosphate buffered saline (PBSA). Each mouse receives 5 x 10 ⁵ cells in 0.1 ml suspension subcutaneously (SC) in the right flank. Tumors are measured in two dimensions to monitor growth when the average volume reaches 80-120 mm ³ . Volume (V) = ½ L × W ² and L (length) is defined as the largest diameter of the tumor and W (width) perpendicular to L. On day 1 of the study, animals are sorted into seven groups (n=10 per group) with an average tumor volume of group 100 mm ³ and treated as described below. Tumor measurements are recorded 2-3 times per week until the tumor volume reaches more than 1200 mm ³ .

Quantitative PCR (qPCR) gene expression analysis was performed on 30 mg of harvested tumor tissue homogenized in 900 μl of lysis buffer supplied in the RNeasy Plus Mini Kit using an Omin Bead Ruptor. RNA was isolated from homogenized tumor samples using the RNeasy Plus Mini Kit and manufacturer-recommended protocols. RNA concentration was quantified using an Epoch BioTek spectrophotometer and resuspended to 200 ng/μl with ddH20. cDNA was synthesized using 2 μg of total RNA and a high-capacity cDNA reverse transcription kit using protocols recommended by the manufacturer. Gene expression was analyzed using 50 ng cDNA and taqman gene-specific primers, TaqMan Universal Master Mix II and protocols recommended by the supplier. The ViiA7 qRT-PCR system is used for qPCR studies. Threshold cycles (CT) for each sample were analyzed using the recommended comparative CT method, and target gene expression was reported as fold change in the treatment group compared to the isotype control group. A two-tailed unpaired Student's t test is used to compare the treatment group with the isotype control group with statistical significance <0.05.

Results:

To study the effect of αvβ8 blockade on tumor lymphocyte numbers, tumor tissue was collected on day 12 (antibody treatment on days 1, 5, 9) from mice treated with isotype control or ADWA11 VH05-2/VK01 (ADWA 2.4), and mRNA expression was analyzed lymphocyte marker using quantitative PCR. Treatment with ADWA11 VH05-2/VK01 (ADWA 2.4) increases the expression levels of CD8a (2.62 ± 0.763) and Granzyme B (5.79 ± 1.55) in the tumor microenvironment (fold change ± standard deviation compared with isotype group, group treatment 10 mg/kg) (FIG. 31A). These data demonstrate that treatment with ADWA11 2.4 antibody increases the number of activated lymphocytes, which play a role in tumor regression, in the tumor microenvironment.

Treatment with ADWA11 2.4 produced an 18% TRI on study day 15, while treatment with anti-PD-1 antibody (RMP1-14) produced a 10.3% TRI on study day 16, and no mice reached the end of the study (0 survival % on day 30). In comparison, ADWA11 2.4 in combination with anti-PD-1 produced 35.8% TPO at day 16 of the study and 60% of mice reached the end of the study (% survival at day 30) (FIG. 31B top panel).

These results demonstrate that combination therapy of anti-PD1 and anti-αvβ8, as demonstrated by the antibody used herein, produces an unexpected synergistic antitumor effect. These data indicate that combination therapy with a novel anti-αvβ8 (eg, ADWA11 2.4) antibody described herein and an anti-PD-1 antibody well known in the art provides a potentially new therapeutic agent for the treatment of tumors.

Although the disclosed teachings have been described with reference to various applications, methods, kits and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein and the claims below. The above examples are presented to better illustrate the ideas disclosed and are not intended to limit the scope of the ideas presented herein. Although the present teachings have been described in terms of these exemplary embodiments, one skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present ideas.

All references cited herein, including patents, patent applications, articles, textbooks and the like, and references cited herein, to the extent not already cited, are hereby incorporated by reference in their entirety. for all purposes. To the extent that one or more of the included literature or similar materials differs from or is inconsistent with this application, including, but not limited to, certain terms, usage of terms, methods described, and the like, this application controls.

It will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope and spirit of the invention. Other embodiments as described will be apparent to those skilled in the art from a consideration of the description and practice of the invention herein. It is intended that the description and examples be considered as illustrative only, the true scope and spirit of the invention being set forth in the following claims.

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LIST OF SEQUENCES

<110> PFIZER INC.

THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

<120> ANTI-αvβ8 ANTIBODIES AND COMPOSITIONS AND THEIR APPLICATION

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Glu Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 4

<211> 714

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 4

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggcgt gcactccgac

60

atccagatga cccagtcccc ttccagcctg agcgcttccg tgggcgacag ggtgaccatc

120

acctgcaggt ccaccaagtc cctgtcccac ttcaacggca acacctacct gttctggtac

180

cagcagaagc ccggcaaggc ccccaagagg ctgatctact acatgtcctc cctggcctcc

240

ggagtgccct ccaggttctc cggatccggc tccggcaccg acttcaccct gaccatctcc

300

tccctgcagc ccgaggattt cgccacctac tactgccagc agtccctgga gtaccccttc

360

accttcggcg gcggcaccaa ggtggagatc aaacgaactg tggctgcacc atctgtcttc

420

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg

480

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg

540

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc

600

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc

660

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt

714

<210> 5

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Ser His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Ser Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 6

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Glu Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 7

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 7

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Ser His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Ser Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 8

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 8

Asp Tyr Tyr Met Asn

15

<210> 9

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 9

Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Glu Pro Lys Phe Gln

1 5 10 15

Gly

<210> 10

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 10

Arg Leu Leu Met Asp Tyr

15

<210> 11

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 11

Arg Ser Thr Lys Ser Leu Ser His Phe Asn Gly Asn Thr Tyr Leu Phe

1 5 10 15

<210> 12

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 12

Tyr Tyr Met Ser Ser Leu Ala Ser

15

<210> 13

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 13

Gln Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 14

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 14

Gly Phe Asn Ile Lys Asp Tyr Tyr Met Asn

1 5 10

<210> 15

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 15

Trp Ile Asp Pro Asp Gln Gly Asn

15

<210> 16

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 16

Arg Leu Leu Met Asp Tyr

15

<210> 17

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 17

Ser Thr Lys Ser Leu Ser His Phe Asn Gly Asn Thr Tyr Leu

1 5 10

<210> 18

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 18

Tyr Tyr Met Ser Ser

15

<210> 19

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 19

Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 20

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 20

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Phe Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Leu Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser

115

<210> 21

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 21

Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 22

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 22

Asp Tyr Tyr Met Asn

15

<210> 23

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 23

Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 24

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 24

Arg Leu Leu Met Asp Tyr

15

<210> 25

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 25

Arg Ser Thr Lys Ser Leu Leu His Phe Asn Gly Asn Thr Tyr Leu Phe

1 5 10 15

<210> 26

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 26

Tyr Tyr Met Ser Asn Leu Ala Ser

15

<210> 27

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 27

Met Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 28

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 28

Gly Phe Asn Ile Lys Asp Tyr Tyr Met Asn

1 5 10

<210> 29

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 29

Trp Ile Asp Pro Asp Asn Gly Asn

15

<210> 30

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 30

Arg Leu Leu Met Asp Tyr

15

<210> 31

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 31

Ser Thr Lys Ser Leu Leu His Phe Asn Gly Asn Thr Tyr Leu

1 5 10

<210> 32

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 32

Tyr Tyr Met Ser Asn

15

<210> 33

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 33

Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 34

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 35

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 35

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 36

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 36

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 37

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 38

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 39

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 40

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Glu Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 41

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 42

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 43

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ala Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 44

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Gln Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 45

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Ala Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 46

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Ala Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 47

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 48

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Ile Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 49

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Ser His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 50

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Ser Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 51

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 51

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Ala Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 52

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 53

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Ser Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 54

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 54

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Gln Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 55

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 55

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 56

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 56

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Tyr Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 57

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 58

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 59

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 60

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 60

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 61

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 62

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 62

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 63

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 63

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 64

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 64

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 65

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 65

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 66

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 66

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Ile Pro

50 55 60

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 67

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Ser His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 68

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 68

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Ser His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Tyr Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 69

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 69

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Ser His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 70

<211> 325

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 70

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro

100 105 110

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

115 120 125

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

130 135 140

Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

145 150 155 160

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

165 170 175

Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu

180 185 190

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala

195 200 205

Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro

210 215 220

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

225 230 235 240

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

245 250 255

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

260 265 270

Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

275 280 285

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

290 295 300

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

305 310 315 320

Leu Ser Pro Gly Lys

325

<210> 71

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 71

Arg Ser Thr Lys Ser Leu Leu His Phe Asn Gly Asn Thr Tyr Leu Phe

1 5 10 15

<210> 72

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 72

Tyr Tyr Met Ser Asn Leu Ala Ser

15

<210> 73

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 73

Met Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 74

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 74

Ser Thr Lys Ser Leu Leu His Phe Asn Gly Asn Thr Tyr Leu

1 5 10

<210> 75

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 75

Tyr Tyr Met Ser Asn

15

<210> 76

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 76

Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 77

<211> 1002

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 77

Met Leu Leu Gly Thr Leu Leu Leu Ile Leu Tyr Ile Leu Met Leu Cys

1 5 10 15

Arg Met Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro

20 25 30

Gly Ile Val Glu Gly Gly Gln Val Leu Lys Cys Asp Trp Ser Ser Thr

35 40 45

Arg Arg Cys Gln Pro Ile Glu Phe Asp Ala Thr Gly Asn Arg Asp Tyr

50 55 60

Ala Lys Asp Asp Pro Leu Glu Phe Lys Ser His Gln Trp Phe Gly Ala

65 70 75 80

Ser Val Arg Ser Lys Gln Asp Lys Ile Leu Ala Cys Ala Pro Leu Tyr

85 90 95

His Trp Arg Thr Glu Met Lys Gln Glu Arg Glu Pro Val Gly Thr Cys

100 105 110

Phe Leu Gln Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser

115 120 125

Gln Asp Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser

130 135 140

Ile Asp Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser

145 150 155 160

Phe Tyr Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu Ile Val

165 170 175

Ser Lys Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr Asn Asn Gln Leu

180 185 190

Ala Thr Arg Thr Ala Gln Ala Ile Phe Asp Asp Ser Tyr Leu Gly Tyr

195 200 205

Ser Val Ala Val Gly Asp Phe Asn Gly Asp Gly Ile Asp Asp Phe Val

210 215 220

Ser Gly Val Pro Arg Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr

225 230 235 240

Asp Gly Lys Asn Met Ser Ser Leu Tyr Asn Phe Thr Gly Glu Gln Met

245 250 255

Ala Ala Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp

260 265 270

Asp Tyr Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp Arg Gly

275 280 285

Ser Asp Gly Lys Leu Gln Glu Val Gly Gln Val Ser Val Ser Leu Gln

290 295 300

Arg Ala Ser Gly Asp Phe Gln Thr Thr Lys Leu Asn Gly Phe Glu Val

305 310 315 320

Phe Ala Arg Phe Gly Ser Ala Ile Ala Pro Leu Gly Asp Leu Asp Gln

325 330 335

Asp Gly Phe Asn Asp Ile Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp

340 345 350

Lys Lys Gly Ile Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn

355 360 365

Ala Val Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Arg Ser Met

370 375 380

Pro Pro Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Ile Asp Lys

385 390 395 400

Asn Gly Tyr Pro Asp Leu Ile Val Gly Ala Phe Gly Val Asp Arg Ala

405 410 415

Ile Leu Tyr Arg Ala Arg Pro Val Ile Thr Val Asn Ala Gly Leu Glu

420 425 430

Val Tyr Pro Ser Ile Leu Asn Gln Asp Asn Lys Thr Cys Ser Leu Pro

435 440 445

Gly Thr Ala Leu Lys Val Ser Cys Phe Asn Val Arg Phe Cys Leu Lys

450 455 460

Ala Asp Gly Lys Gly Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu

465 470 475 480

Leu Leu Leu Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu

485 490 495

Phe Leu Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr Ile Ser

500 505 510

Arg Gly Gly Leu Met Gln Cys Glu Glu Leu Ile Ala Tyr Leu Arg Asp

515 520 525

Glu Ser Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr Ile Phe Met Glu

530 535 540

Tyr Arg Leu Asp Tyr Arg Thr Ala Ala Asp Thr Thr Gly Leu Gln Pro

545 550 555 560

Ile Leu Asn Gln Phe Thr Pro Ala Asn Ile Ser Arg Gln Ala His Ile

565 570 575

Leu Leu Asp Cys Gly Glu Asp Asn Val Cys Lys Pro Lys Leu Glu Val

580 585 590

Ser Val Asp Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro

595 600 605

Leu Thr Leu Ile Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu

610 615 620

Ala Glu Leu Ile Val Ser Ile Pro Leu Gln Ala Asp Phe Ile Gly Val

625 630 635 640

Val Arg Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe Lys Thr

645 650 655

Glu Asn Gln Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met Lys

660 665 670

Ala Gly Thr Gln Leu Leu Ala Gly Leu Arg Phe Ser Val His Gln Gln

675 680 685

Ser Glu Met Asp Thr Ser Val Lys Phe Asp Leu Gln Ile Gln Ser Ser

690 695 700

Asn Leu Phe Asp Lys Val Ser Pro Val Val Ser His Lys Val Asp Leu

705 710 715 720

Ala Val Leu Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His

725 730 735

Ile Phe Leu Pro Ile Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr

740 745 750

Glu Glu Asp Val Gly Pro Val Val Gln His Ile Tyr Glu Leu Arg Asn

755 760 765

Asn Gly Pro Ser Ser Phe Ser Lys Ala Met Leu His Leu Gln Trp Pro

770 775 780

Tyr Lys Tyr Asn Asn Asn Thr Leu Leu Tyr Ile Leu His Tyr Asp Ile

785 790 795 800

Asp Gly Pro Met Asn Cys Thr Ser Asp Met Glu Ile Asn Pro Leu Arg

805 810 815

Ile Lys Ile Ser Ser Leu Gln Thr Thr Glu Lys Asn Asp Thr Val Ala

820 825 830

Gly Gln Gly Glu Arg Asp His Leu Ile Thr Lys Arg Asp Leu Ala Leu

835 840 845

Ser Glu Gly Asp Ile His Thr Leu Gly Cys Gly Val Ala Gln Cys Leu

850 855 860

Lys Ile Val Cys Gln Val Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile

865 870 875 880

Leu Tyr Val Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn Lys Glu

885 890 895

Asn Gln Asn His Ser Tyr Ser Leu Lys Ser Ser Ala Ser Phe Asn Val

900 905 910

Ile Glu Phe Pro Tyr Lys Asn Leu Pro Ile Glu Asp Ile Thr Asn Ser

915 920 925

Thr Leu Val Thr Thr Asn Val Thr Trp Gly Ile Gln Pro Ala Pro Met

930 935 940

Pro Val Pro Val Trp Val Ile Ile Leu Ala Val Leu Ala Gly Leu Leu

945 950 955 960

Leu Leu Ala Val Leu Val Phe Val Met Tyr Arg Met Gly Phe Phe Lys

965 970 975

Arg Val Arg Pro Pro Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln Pro

980 985 990

His Glu Asn Gly Glu Gly Asn Ser Glu Thr

995 1000

<210> 78

<211> 769

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 78

Met Cys Gly Ser Ala Leu Ala Phe Phe Thr Ala Ala Phe Val Cys Leu

1 5 10 15

Gln Asn Asp Arg Arg Gly Pro Ala Ser Phe Leu Trp Ala Ala Trp Val

20 25 30

Phe Ser Leu Val Leu Gly Leu Gly Gln Gly Glu Asp Asn Arg Cys Ala

35 40 45

Ser Ser Asn Ala Ala Ser Cys Ala Arg Cys Leu Ala Leu Gly Pro Glu

50 55 60

Cys Gly Trp Cys Val Gln Glu Asp Phe Ile Ser Gly Gly Ser Arg Ser

65 70 75 80

Glu Arg Cys Asp Ile Val Ser Asn Leu Ile Ser Lys Gly Cys Ser Val

85 90 95

Asp Ser Ile Glu Tyr Pro Ser Val His Val Ile Ile Pro Thr Glu Asn

100 105 110

Glu Ile Asn Thr Gln Val Thr Pro Gly Glu Val Ser Ile Gln Leu Arg

115 120 125

Pro Gly Ala Glu Ala Asn Phe Met Leu Lys Val His Pro Leu Lys Lys

130 135 140

Tyr Pro Val Asp Leu Tyr Tyr Leu Val Asp Val Ser Ala Ser Met His

145 150 155 160

Asn Asn Ile Glu Lys Leu Asn Ser Val Gly Asn Asp Leu Ser Arg Lys

165 170 175

Met Ala Phe Phe Ser Arg Asp Phe Arg Leu Gly Phe Gly Ser Tyr Val

180 185 190

Asp Lys Thr Val Ser Pro Tyr Ile Ser Ile His Pro Glu Arg Ile His

195 200 205

Asn Gln Cys Ser Asp Tyr Asn Leu Asp Cys Met Pro Pro His Gly Tyr

210 215 220

Ile His Val Leu Ser Leu Thr Glu Asn Ile Thr Glu Phe Glu Lys Ala

225 230 235 240

Val His Arg Gln Lys Ile Ser Gly Asn Ile Asp Thr Pro Glu Gly Gly

245 250 255

Phe Asp Ala Met Leu Gln Ala Ala Val Cys Glu Ser His Ile Gly Trp

260 265 270

Arg Lys Glu Ala Lys Arg Leu Leu Leu Val Met Thr Asp Gln Thr Ser

275 280 285

His Leu Ala Leu Asp Ser Lys Leu Ala Gly Ile Val Val Pro Asn Asp

290 295 300

Gly Asn Cys His Leu Lys Asn Asn Val Tyr Val Lys Ser Thr Thr Met

305 310 315 320

Glu His Pro Ser Leu Gly Gln Leu Ser Glu Lys Leu Ile Asp Asn Asn

325 330 335

Ile Asn Val Ile Phe Ala Val Gln Gly Lys Gln Phe His Trp Tyr Lys

340 345 350

Asp Leu Leu Pro Leu Leu Pro Gly Thr Ile Ala Gly Glu Ile Glu Ser

355 360 365

Lys Ala Ala Asn Leu Asn Asn Leu Val Val Glu Ala Tyr Gln Lys Leu

370 375 380

Ile Ser Glu Val Lys Val Gln Val Glu Asn Gln Val Gln Gly Ile Tyr

385 390 395 400

Phe Asn Ile Thr Ala Ile Cys Pro Asp Gly Ser Arg Lys Pro Gly Met

405 410 415

Glu Gly Cys Arg Asn Val Thr Ser Asn Asp Glu Val Leu Phe Asn Val

420 425 430

Thr Val Thr Met Lys Lys Cys Asp Val Thr Gly Gly Lys Asn Tyr Ala

435 440 445

Ile Ile Lys Pro Ile Gly Phe Asn Glu Thr Ala Lys Ile His Ile His

450 455 460

Arg Asn Cys Ser Cys Gln Cys Glu Asp Asn Arg Gly Pro Lys Gly Lys

465 470 475 480

Cys Val Asp Glu Thr Phe Leu Asp Ser Lys Cys Phe Gln Cys Asp Glu

485 490 495

Asn Lys Cys His Phe Asp Glu Asp Gln Phe Ser Ser Glu Ser Cys Lys

500 505 510

Ser His Lys Asp Gln Pro Val Cys Ser Gly Arg Gly Val Cys Val Cys

515 520 525

Gly Lys Cys Ser Cys His Lys Ile Lys Leu Gly Lys Val Tyr Gly Lys

530 535 540

Tyr Cys Glu Lys Asp Asp Phe Ser Cys Pro Tyr His His Gly Asn Leu

545 550 555 560

Cys Ala Gly His Gly Glu Cys Glu Ala Gly Arg Cys Gln Cys Phe Ser

565 570 575

Gly Trp Glu Gly Asp Arg Cys Gln Cys Pro Ser Ala Ala Ala Gln His

580 585 590

Cys Val Asn Ser Lys Gly Gln Val Cys Ser Gly Arg Gly Thr Cys Val

595 600 605

Cys Gly Arg Cys Glu Cys Thr Asp Pro Arg Ser Ile Gly Arg Phe Cys

610 615 620

Glu His Cys Pro Thr Cys Tyr Thr Ala Cys Lys Glu Asn Trp Asn Cys

625 630 635 640

Met Gln Cys Leu His Pro His Asn Leu Ser Gln Ala Ile Leu Asp Gln

645 650 655

Cys Lys Thr Ser Cys Ala Leu Met Glu Gln Gln His Tyr Val Asp Gln

660 665 670

Thr Ser Glu Cys Phe Ser Ser Pro Ser Tyr Leu Arg Ile Phe Phe Ile

675 680 685

Ile Phe Ile Val Thr Phe Leu Ile Gly Leu Leu Lys Val Leu Ile Ile

690 695 700

Arg Gln Val Ile Leu Gln Trp Asn Ser Asn Lys Ile Lys Ser Ser Ser

705 710 715 720

Asp Tyr Arg Val Ser Ala Ser Lys Lys Asp Lys Leu Ile Leu Gln Ser

725 730 735

Val Cys Thr Arg Ala Val Thr Tyr Arg Arg Glu Lys Pro Glu Glu Ile

740 745 750

Lys Met Asp Ile Ser Lys Leu Asn Ala His Glu Thr Phe Arg Cys Asn

755 760 765

Phe

<210> 79

<211> 1044

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 79

Met Ala Ala Pro Gly Arg Leu Leu Leu Arg Pro Arg Pro Gly Gly Leu

1 5 10 15

Leu Leu Leu Leu Pro Gly Leu Leu Leu Pro Leu Ala Asp Ala Phe Asn

20 25 30

Leu Asp Val Glu Ser Pro Ala Glu Tyr Ala Gly Pro Glu Gly Ser Tyr

35 40 45

Phe Gly Phe Ala Val Asp Phe Phe Glu Pro Ser Thr Ser Ser Arg Met

50 55 60

Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro Gly Ile

65 70 75 80

Val Glu Gly Gly Gln Val Leu Lys Cys Glu Cys Ser Ser Ser Arg Arg

85 90 95

Cys Gln Pro Ile Glu Phe Asp Ser Thr Gly Asn Arg Asp Tyr Ala Lys

100 105 110

Asp Asp Pro Leu Glu Phe Lys Ser His Gln Trp Phe Gly Ala Ser Val

115 120 125

Arg Ser Lys Gln Asp Lys Ile Leu Ala Cys Ala Pro Leu Tyr His Trp

130 135 140

Arg Thr Glu Met Lys Gln Glu Arg Glu Pro Val Gly Thr Cys Phe Leu

145 150 155 160

Gln Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser Lys Asn

165 170 175

Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser Ile Asp

180 185 190

Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser Phe Tyr

195 200 205

Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu Ile Ile Ser Lys

210 215 220

Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr Asn Asn Gln Leu Ala Thr

225 230 235 240

Arg Thr Ala Gln Ala Ile Phe Asp Asp Ser Tyr Leu Gly Tyr Ser Val

245 250 255

Ala Val Gly Asp Phe Asn Gly Asp Gly Ile Glu Asp Phe Val Ser Gly

260 265 270

Val Pro Arg Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr Asp Gly

275 280 285

Lys Asn Met Ser Ser Leu His Asn Phe Thr Gly Glu Gln Met Ala Ala

290 295 300

Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp Asp Tyr

305 310 315 320

Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp Arg Gly Ser Asp

325 330 335

Gly Lys Leu Gln Glu Val Gly Gln Val Ser Val Ser Leu Gln Arg Ala

340 345 350

Val Gly Asp Phe Gln Thr Thr Lys Leu Asn Gly Phe Glu Val Phe Ala

355 360 365

Arg Phe Gly Ser Ala Ile Ala Pro Leu Gly Asp Leu Asp Gln Asp Gly

370 375 380

Phe Asn Asp Ile Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp Lys Lys

385 390 395 400

Gly Leu Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn Ser Val

405 410 415

Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Gln Ser Met Pro Pro

420 425 430

Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Val Asp Arg Asn Gly

435 440 445

Tyr Pro Asp Leu Val Val Gly Ala Phe Gly Val Asp Arg Ala Val Leu

450 455 460

Tyr Arg Ala Arg Pro Val Val Thr Val Asn Ala Gly Leu Glu Val Tyr

465 470 475 480

Pro Ser Ile Leu Asn Gln Asp Asn Lys Ile Cys Pro Leu Pro Gly Thr

485 490 495

Ala Leu Lys Val Ser Cys Phe Asn Val Arg Phe Cys Leu Lys Ala Asp

500 505 510

Gly Lys Gly Thr Leu Pro Arg Lys Leu His Phe Gln Val Glu Leu Leu

515 520 525

Leu Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu Phe Leu

530 535 540

His Asn Arg Ser Pro Val His Ser Lys Thr Met Thr Val Phe Arg Gly

545 550 555 560

Gly Gln Met Gln Cys Glu Glu Leu Val Ala Tyr Leu Arg Asp Glu Ser

565 570 575

Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr Ile Phe Met Glu Tyr Arg

580 585 590

Leu Asp Gln Arg Thr Ala Ala Asp Ala Thr Gly Leu Gln Pro Ile Leu

595 600 605

Asn Gln Phe Thr Pro Ala Asn Val Ser Arg Gln Ala His Ile Leu Leu

610 615 620

Asp Cys Gly Glu Asp Asn Val Cys Lys Pro Lys Leu Glu Val Ser Val

625 630 635 640

Asn Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro Leu Thr

645 650 655

Leu Thr Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu Ala Glu

660 665 670

Leu Ile Val Ser Ile Pro Pro Gln Ala Asp Phe Ile Gly Val Val Arg

675 680 685

Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe Lys Thr Glu Asn

690 695 700

Gln Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met Lys Ala Gly

705 710 715 720

Thr Gln Leu Leu Ala Gly Leu Arg Phe Ser Val His Gln Gln Ser Glu

725 730 735

Met Asp Thr Ser Val Lys Phe Asp Leu Lys Ile Gln Ser Ser Asn Ser

740 745 750

Phe Asp Asn Val Ser Pro Val Val Ser Tyr Lys Val Asp Leu Ala Val

755 760 765

Leu Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His Ile Phe

770 775 780

Leu Pro Ile Pro Asn Trp Glu Tyr Lys Glu Asn Pro Glu Thr Glu Glu

785 790 795 800

Asp Val Gly Pro Ile Val Gln His Ile Tyr Glu Leu Arg Asn Asn Gly

805 810 815

Pro Ser Ser Phe Ser Lys Ala Ile Leu Asn Leu Gln Trp Pro Tyr Lys

820 825 830

Tyr Asn Asn Asn Thr Leu Leu Tyr Ile Leu His Tyr Asp Ile Asp Gly

835 840 845

Pro Met Asn Cys Thr Ala Asp Thr Glu Ile Asn Pro Leu Arg Ile Lys

850 855 860

Thr Pro Glu Lys Asn Asp Thr Ala Ala Ala Gly Gln Gly Glu Arg Asn

865 870 875 880

His Leu Ile Thr Lys Arg Asp Leu Thr Leu Arg Glu Gly Asp Val His

885 890 895

Thr Leu Gly Cys Gly Ile Ala Lys Cys Leu Gln Ile Thr Cys Gln Val

900 905 910

Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile Leu Tyr Val Lys Ser Leu

915 920 925

Leu Trp Thr Glu Thr Phe Met Asn Lys Glu Asn Gln Asn His Ser Tyr

930 935 940

Ser Leu Lys Ser Ser Ala Ser Phe Asn Ile Ile Glu Phe Pro Tyr Lys

945 950 955 960

Asn Leu Pro Ile Glu Asp Leu Phe Asn Ser Thr Leu Val Thr Thr Asn

965 970 975

Ile Thr Trp Gly Ile Gln Pro Ala Pro Met Pro Val Pro Val Trp Val

980 985 990

Ile Ile Leu Ala Val Leu Ala Gly Leu Leu Leu Leu Ala Val Leu Val

995 1000 1005

Phe Val Met Tyr Arg Met Gly Phe Phe Lys Arg Val Arg Pro Pro

1010 1015 1020

Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln Pro His Glu Asn Gly

1025 1030 1035

Glu Gly Asn Ser Glu Thr

1040

<210> 80

<211> 767

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 80

Met Cys Gly Ser Ala Leu Ala Phe Leu Thr Ala Ala Leu Leu Ser Leu

1 5 10 15

His Asn Cys Gln Arg Gly Pro Ala Leu Val Leu Gly Ala Ala Trp Val

20 25 30

Phe Ser Leu Val Leu Gly Leu Gly Gln Ser Glu His Asn Arg Cys Gly

35 40 45

Ser Ala Asn Val Val Ser Cys Ala Arg Cys Leu Gln Leu Gly Pro Glu

50 55 60

Cys Gly Trp Cys Val Gln Glu Asp Phe Val Ser Gly Gly Ser Gly Ser

65 70 75 80

Glu Arg Cys Asp Thr Val Ser Ser Leu Ile Ser Lys Gly Cys Pro Val

85 90 95

Asp Ser Ile Glu Tyr Leu Ser Val His Val Val Thr Ser Ser Glu Asn

100 105 110

Glu Ile Asn Thr Gln Val Thr Pro Gly Glu Val Ser Val Gln Leu His

115 120 125

Pro Gly Ala Glu Ala Asn Phe Met Leu Lys Val Arg Pro Leu Lys Lys

130 135 140

Tyr Pro Val Asp Leu Tyr Tyr Leu Val Asp Val Ser Ala Ser Met His

145 150 155 160

Asn Asn Ile Glu Lys Leu Asn Ser Val Gly Asn Asp Leu Ser Lys Lys

165 170 175

Met Ala Leu Tyr Ser Arg Asp Phe Arg Leu Gly Phe Gly Ser Tyr Val

180 185 190

Asp Lys Thr Val Ser Pro Tyr Ile Ser Ile His Pro Glu Arg Ile His

195 200 205

Asn Gln Cys Ser Asp Tyr Asn Leu Asp Cys Met Pro Pro His Gly Tyr

210 215 220

Ile His Val Leu Ser Leu Thr Glu Asn Ile Thr Glu Phe Glu Lys Ala

225 230 235 240

Val His Arg Gln Lys Ile Ser Gly Asn Ile Asp Thr Pro Glu Gly Gly

245 250 255

Phe Asp Ala Met Leu Gln Ala Ala Val Cys Glu Ser His Ile Gly Trp

260 265 270

Arg Lys Glu Ala Lys Arg Leu Leu Leu Val Met Thr Asp Gln Thr Ser

275 280 285

His Leu Ala Leu Asp Ser Lys Leu Ala Gly Ile Val Val Pro Asn Asp

290 295 300

Gly Asn Cys His Leu Lys Asn Asn Val Tyr Val Lys Ser Thr Thr Met

305 310 315 320

Glu His Pro Ser Leu Gly Gln Leu Ser Glu Lys Leu Ile Asp Asn Asn

325 330 335

Ile Asn Val Ile Phe Ala Val Gln Gly Lys Gln Phe His Trp Tyr Lys

340 345 350

Asp Leu Leu Pro Leu Leu Pro Gly Ala Ile Ala Gly Glu Ile Glu Ser

355 360 365

Lys Ala Ala Asn Leu Asn Asn Leu Val Val Glu Ala Tyr Lys Lys Ile

370 375 380

Ile Ser Glu Val Lys Val Gln Leu Glu Asn Gln Val His Gly Val His

385 390 395 400

Phe Asn Ile Thr Ala Ile Cys Pro Asp Gly Ala Arg Lys Pro Gly Ile

405 410 415

Ser Gly Cys Gly Asn Val Thr Ser Asn Asp Glu Val Leu Phe Asn Val

420 425 430

Thr Val Val Met Lys Thr Cys Asp Ile Met Gly Gly Lys Asn Tyr Ala

435 440 445

Ile Ile Lys Pro Ile Gly Phe Asn Glu Thr Thr Lys Val His Ile His

450 455 460

Arg Ser Cys Ser Cys Gln Cys Glu Asn His Arg Gly Leu Lys Gly Gln

465 470 475 480

Cys Ala Glu Ala Ala Pro Asp Pro Lys Cys Pro Gln Cys Asp Asp Ser

485 490 495

Arg Cys His Phe Asp Glu Asp Gln Phe Pro Ser Glu Thr Cys Lys Pro

500 505 510

Gln Glu Asp Gln Pro Val Cys Ser Gly Arg Gly Val Cys Ile Cys Gly

515 520 525

Lys Cys Leu Cys His Lys Thr Lys Leu Gly Arg Val Tyr Gly Gln Tyr

530 535 540

Cys Glu Lys Asp Asp Phe Ser Cys Pro Tyr Leu His Gly Asp Val Cys

545 550 555 560

Ala Gly His Gly Glu Cys Glu Gly Gly Arg Cys Gln Cys Phe Ser Gly

565 570 575

Trp Glu Gly Asp Arg Cys Gln Cys Pro Ser Ala Ser Ala Gln His Cys

580 585 590

Val Asn Ser Lys Gly Gln Val Cys Ser Gly Arg Gly Thr Cys Val Cys

595 600 605

Gly Arg Cys Glu Cys Thr Asp Pro Arg Ser Ile Gly Arg Leu Cys Glu

610 615 620

His Cys Pro Thr Cys His Leu Ser Cys Ser Glu Asn Trp Asn Cys Leu

625 630 635 640

Gln Cys Leu His Pro His Asn Leu Ser Gln Ala Ala Leu Asp Gln Cys

645 650 655

Lys Ser Ser Cys Ala Val Met Glu Gln His Arg Met Asp Gln Thr Ser

660 665 670

Glu Cys Leu Ser Gly Pro Ser Tyr Leu Arg Ile Phe Phe Ile Ile Phe

675 680 685

Ile Val Thr Phe Leu Ile Gly Leu Leu Lys Val Leu Ile Ile Arg Gln

690 695 700

Val Ile Leu Gln Trp Asn Asn Asn Lys Ile Lys Ser Ser Ser Asp Tyr

705 710 715 720

Arg Met Ser Ala Ser Lys Lys Asp Lys Leu Ile Leu Gln Ser Val Cys

725 730 735

Thr Arg Ala Val Thr Tyr Arg Arg Glu Lys Pro Glu Glu Ile Lys Met

740 745 750

Asp Ile Ser Lys Leu Asn Ala Gln Glu Ala Phe Arg Cys Asn Phe

755 760 765

<210> 81

<211> 329

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 81

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 82

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 82

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 83

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 83

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 84

<211> 1048

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 84

Met Ala Ser Pro Pro Arg Arg Arg Leu Arg Leu Gly Pro Arg Gly Leu

1 5 10 15

Pro Leu Leu Leu Ser Gly Leu Leu Leu Pro Leu Cys Arg Ala Phe Asn

20 25 30

Leu Asp Val Asp Ser Pro Ala Glu Tyr Ser Gly Pro Glu Gly Ser Tyr

35 40 45

Phe Gly Phe Ala Val Asp Phe Phe Val Pro Ser Ala Ser Ser Arg Met

50 55 60

Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro Gly Ile

65 70 75 80

Val Glu Gly Gly Gln Val Leu Lys Cys Asp Trp Ser Ser Thr Arg Arg

85 90 95

Cys Gln Pro Ile Glu Phe Asp Ala Thr Gly Asn Arg Asp Tyr Ala Lys

100 105 110

Asp Asp Pro Leu Glu Phe Lys Ser His Gln Trp Phe Gly Ala Ser Val

115 120 125

Arg Ser Lys Gln Asp Lys Ile Leu Ala Cys Ala Pro Leu Tyr His Trp

130 135 140

Arg Thr Glu Leu Lys Gln Glu Arg Glu Pro Val Gly Thr Cys Phe Leu

145 150 155 160

Gln Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser Gln Asp

165 170 175

Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser Ile Asp

180 185 190

Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser Phe Tyr

195 200 205

Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu Ile Val Ser Lys

210 215 220

Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr Asn Asn Gln Leu Ala Thr

225 230 235 240

Arg Thr Ala Gln Ala Ile Phe Asp Asp Ser Tyr Leu Gly Tyr Ser Val

245 250 255

Ala Val Gly Asp Phe Asn Gly Asp Gly Ile Asp Asp Phe Val Ser Gly

260 265 270

Val Pro Arg Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr Asp Gly

275 280 285

Lys Asn Met Ser Ser Ile Tyr Asn Phe Thr Gly Asp Gln Met Ala Ala

290 295 300

Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp Asp Tyr

305 310 315 320

Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp Arg Gly Ser Asp

325 330 335

Gly Lys Leu Gln Glu Val Gly Gln Val Ser Val Ser Leu Gln Arg Ala

340 345 350

Ser Gly Asp Phe Gln Thr Thr Lys Leu Asn Gly Phe Glu Val Phe Ala

355 360 365

Arg Phe Gly Ser Ala Ile Ala Pro Leu Gly Asp Leu Asp Gln Asp Gly

370 375 380

Phe Asn Asp Ile Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp Lys Lys

385 390 395 400

Gly Ile Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn Ala Val

405 410 415

Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Arg Ser Met Pro Pro

420 425 430

Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Ile Asp Lys Asn Gly

435 440 445

Tyr Pro Asp Leu Ile Val Gly Ala Phe Gly Val Asp Arg Ala Ile Leu

450 455 460

Tyr Arg Ala Arg Pro Val Ile Thr Val Asn Ala Gly Leu Glu Val Tyr

465 470 475 480

Pro Ser Ile Leu Asn Gln Asp Asn Lys Thr Cys Ser Leu Pro Gly Thr

485 490 495

Ala Leu Lys Val Ser Cys Phe Asn Val Arg Phe Cys Leu Lys Ala Asp

500 505 510

Gly Lys Gly Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu Leu Leu

515 520 525

Leu Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu Phe Leu

530 535 540

Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr Ile Ser Arg Gly

545 550 555 560

Gly Leu Met Gln Cys Glu Glu Leu Ile Ala Tyr Leu Arg Asp Glu Ser

565 570 575

Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr Ile Phe Met Glu Tyr Trp

580 585 590

Leu Asp Tyr Arg Thr Ala Ala Asp Thr Thr Gly Leu Gln Pro Ile Leu

595 600 605

Asn Gln Phe Thr Pro Ala Asn Ile Ser Arg Gln Ala His Ile Leu Leu

610 615 620

Asp Cys Gly Glu Asp Asn Val Cys Lys Pro Lys Leu Glu Val Phe Val

625 630 635 640

Asp Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro Leu Thr

645 650 655

Leu Ile Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu Ala Glu

660 665 670

Leu Ile Val Ser Ile Pro Leu Gln Ala Asp Phe Ile Gly Val Val Arg

675 680 685

Asn Ser Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe Lys Thr Glu Asn

690 695 700

Gln Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met Lys Ala Gly

705 710 715 720

Thr Gln Leu Leu Ala Gly Leu Arg Phe Ser Val His Gln Gln Ser Glu

725 730 735

Met Asp Thr Ser Val Lys Phe Asp Leu Gln Ile Gln Ser Ser Asn Leu

740 745 750

Phe Asp Lys Val Ser Pro Val Val Ser His Lys Val Asp Leu Ala Val

755 760 765

Leu Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His Ile Phe

770 775 780

Leu Pro Ile Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr Glu Glu

785 790 795 800

Asp Val Gly Pro Val Val Gln His Ile Tyr Glu Leu Arg Asn Asn Gly

805 810 815

Pro Ser Ser Phe Ser Lys Ala Met Leu His Leu Gln Trp Pro Tyr Lys

820 825 830

Tyr Asn Asn Asn Thr Leu Leu Tyr Ile Leu His Tyr Asp Ile Asp Gly

835 840 845

Pro Met Asn Cys Thr Ser Asp Met Glu Ile Asn Pro Leu Arg Ile Lys

850 855 860

Ile Ser Ser Leu Gln Ala Thr Glu Lys Asn Asp Thr Val Ala Gly Gln

865 870 875 880

Gly Glu Arg Asp His Leu Ile Thr Lys Arg Asp Leu Ala Leu Ser Glu

885 890 895

Gly Asp Ile His Thr Leu Gly Cys Gly Val Ala Gln Cys Leu Lys Ile

900 905 910

Val Cys Gln Val Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile Leu Tyr

915 920 925

Val Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn Lys Glu Asn Gln

930 935 940

Asn His Ser Tyr Ser Leu Lys Ser Ser Ala Ser Phe Asn Val Ile Glu

945 950 955 960

Phe Pro Tyr Lys Asn Leu Pro Ile Glu Asp Ile Thr Asn Ser Thr Leu

965 970 975

Val Thr Thr Asn Val Thr Trp Gly Ile Gln Pro Ala Pro Met Pro Val

980 985 990

Pro Val Trp Val Ile Ile Leu Ala Val Leu Ala Gly Leu Leu Leu Leu

995 1000 1005

Ala Val Leu Val Phe Val Met Tyr Arg Met Gly Phe Phe Lys Arg

1010 1015 1020

Val Arg Pro Pro Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln Pro

1025 1030 1035

His Glu Asn Gly Glu Gly Asn Ser Glu Thr

1040 1045

<210> 85

<211> 769

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 85

Met Cys Gly Ser Ala Leu Ala Phe Phe Thr Ala Ala Phe Val Cys Leu

1 5 10 15

Gln Asn Asp Arg Arg Gly Pro Ala Ser Phe Leu Trp Ala Ala Trp Val

20 25 30

Leu Ser Leu Val Leu Gly Leu Gly Gln Gly Glu Asp Asn Ile Cys Ala

35 40 45

Ser Ser Asn Ala Ala Ser Cys Ala Arg Cys Leu Ala Leu Gly Pro Glu

50 55 60

Cys Gly Trp Cys Val Gln Glu Asp Phe Ile Ser Gly Gly Ser Arg Ser

65 70 75 80

Glu Arg Cys Asp Ile Val Ser Asn Leu Ile Ser Lys Gly Cys Ser Val

85 90 95

Asp Ser Ile Glu Tyr Pro Ser Val His Val Ile Ile Pro Thr Glu Asn

100 105 110

Glu Ile Asn Thr Gln Val Thr Pro Gly Glu Val Ser Ile Gln Leu Arg

115 120 125

Pro Gly Ala Glu Ala Asn Phe Met Leu Lys Ile His Pro Leu Lys Lys

130 135 140

Tyr Pro Val Asp Leu Tyr Tyr Leu Val Asp Val Ser Ala Ser Met His

145 150 155 160

Asn Asn Ile Glu Lys Leu Asn Ser Val Gly Asn Asp Leu Ser Arg Lys

165 170 175

Met Ala Phe Phe Ser Arg Asp Phe Arg Leu Gly Phe Gly Ser Tyr Val

180 185 190

Asp Lys Thr Val Ser Pro Tyr Ile Ser Ile His Pro Glu Arg Ile His

195 200 205

Asn Gln Cys Ser Asp Tyr Asn Leu Asp Cys Met Pro Pro His Gly Tyr

210 215 220

Ile His Val Leu Ser Leu Thr Glu Asn Ile Thr Glu Phe Glu Lys Ala

225 230 235 240

Val His Arg Gln Lys Ile Ser Gly Asn Ile Asp Thr Pro Glu Gly Gly

245 250 255

Phe Asp Ala Met Leu Gln Ala Ala Val Cys Glu Ser His Ile Gly Trp

260 265 270

Arg Lys Glu Ala Lys Arg Leu Leu Leu Val Met Thr Asp Gln Thr Ser

275 280 285

His Leu Ala Leu Asp Ser Lys Leu Ala Gly Ile Val Val Pro Asn Asp

290 295 300

Gly Asn Cys His Leu Lys Asn Asn Val Tyr Val Lys Ser Thr Thr Met

305 310 315 320

Glu His Pro Ser Leu Gly Gln Leu Ser Glu Lys Leu Ile Asp Asn Asn

325 330 335

Ile Asn Val Ile Phe Ala Val Gln Gly Lys Gln Phe His Trp Tyr Lys

340 345 350

Asp Leu Leu Pro Leu Leu Pro Gly Thr Ile Ala Gly Glu Ile Glu Ser

355 360 365

Lys Ala Ala Asn Leu Asn Asn Leu Val Val Glu Ala Tyr Gln Lys Leu

370 375 380

Ile Ser Glu Val Lys Val His Val Glu Asn Gln Val Gln Gly Val Tyr

385 390 395 400

Phe Asn Ile Thr Ala Ile Cys Pro Asp Gly Ser Arg Lys Pro Gly Met

405 410 415

Glu Gly Cys Arg Asn Val Thr Ser Asn His Glu Val Leu Phe Asn Val

420 425 430

Thr Val Thr Met Lys Lys Cys Asp Val Thr Gly Gly Lys Asn Tyr Ala

435 440 445

Ile Ile Lys Pro Ile Gly Phe Asn Glu Thr Ala Lys Ile His Ile His

450 455 460

Arg Asn Cys Ser Cys Gln Cys Glu Asp Asn Arg Gly Pro Lys Gly Lys

465 470 475 480

Cys Val Asp Glu Thr Phe Leu Asp Ser Lys Cys Phe Gln Cys Asp Glu

485 490 495

Asn Lys Cys His Phe Asp Glu Asp Gln Phe Ser Ser Glu Ser Cys Lys

500 505 510

Ser His Lys Asp Gln Pro Val Cys Ser Gly Arg Gly Val Cys Val Cys

515 520 525

Gly Lys Cys Ser Cys His Lys Ile Lys Leu Gly Lys Val Tyr Gly Lys

530 535 540

Tyr Cys Glu Lys Asp Asp Phe Ser Cys Pro Tyr His His Gly Asn Leu

545 550 555 560

Cys Ala Gly His Gly Glu Cys Glu Ala Gly Arg Cys Gln Cys Phe Ser

565 570 575

Gly Trp Glu Gly Asp Arg Cys Gln Cys Pro Ser Ala Ala Ala Gln His

580 585 590

Cys Val Asn Ser Lys Gly Gln Val Cys Ser Gly Arg Gly Thr Cys Val

595 600 605

Cys Gly Arg Cys Glu Cys Thr Asp Pro Arg Ser Ile Gly Arg Phe Cys

610 615 620

Glu His Cys Pro Thr Cys His Thr Ala Cys Lys Glu Asn Trp Asn Cys

625 630 635 640

Val Gln Cys Leu His Pro His Asn Leu Ser Gln Ala Ile Leu Asp Gln

645 650 655

Cys Lys Thr Ser Cys Ala Leu Met Glu Gln Gln His Tyr Val Asp Gln

660 665 670

Thr Ser Glu Cys Phe Ser Ser Pro Ser Tyr Leu Arg Ile Phe Phe Ile

675 680 685

Ile Phe Ile Val Thr Phe Leu Ile Gly Leu Leu Lys Val Leu Ile Ile

690 695 700

Arg Gln Val Ile Leu Gln Trp Asn Ser Asn Lys Ile Lys Ser Ser Ser

705 710 715 720

Asp Tyr Arg Val Ser Ala Ser Lys Lys Asp Lys Leu Ile Leu Gln Ser

725 730 735

Val Cys Thr Arg Ala Val Thr Tyr Arg Arg Glu Lys Pro Glu Glu Ile

740 745 750

Lys Met Asp Ile Ser Lys Leu Asn Ala His Glu Thr Phe Arg Cys Asn

755 760 765

Phe

<210> 86

<211> 324

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 86

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Pro Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Ala Phe Ala Cys Ala Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 87

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 87

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 88

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 89

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 89

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 90

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Asn Ala Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 91

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 91

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 92

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 92

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Phe Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 93

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 93

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Glu Pro Lys Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 94

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 94

Glu Pro Lys Phe Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

1 5 10 15

<210> 95

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 95

Thr Ala Val Tyr Tyr Cys Ala Arg Arg Leu Leu Met Asp Tyr Trp

1 5 10 15

<210> 96

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<400> 96

Thr Ala Val Tyr Tyr Ser Ala Arg Arg Leu Leu Xaa Asp Tyr Trp

1 5 10 15

<210> 97

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 97

Lys Ser Leu Leu His Phe Asn Gly Asn Thr Tyr Leu Phe Trp Tyr

1 5 10 15

<210> 98

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 98

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val

1 5 10 15

<210> 99

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (8)..(8)

<223> Xaa can be any naturally occurring amino acid

<400> 99

Pro Lys Arg Leu Ile Tyr Tyr Xaa Ser Asn Leu Ala Ser Gly Val

1 5 10 15

<210> 100

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 100

Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

1 5 10 15

<210> 101

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 101

Leu Ile Tyr Tyr Xaa Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

1 5 10 15

<210> 102

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 102

Phe Ala Thr Tyr Tyr Cys Met Gln Ser Leu Glu Tyr Pro Phe Thr

1 5 10 15

<210> 103

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 103

Phe Ala Thr Tyr Tyr Ser Xaa Gln Ser Leu Glu Tyr Pro Phe Thr

1 5 10 15

<210> 104

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 104

Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10 15

<210> 105

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 105

Glu Pro Lys Val Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

1 5 10 15

<210> 106

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 106

Lys Ser Leu Ser His Phe Asn Gly Asn Thr Tyr Leu Phe Trp Tyr

1 5 10 15

<210> 107

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 107

Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Glu Tyr Pro Phe Thr

1 5 10 15

<210> 108

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 108

Phe Ala Thr Tyr Tyr Ser Gln Gln Ser Leu Glu Tyr Pro Phe Thr

1 5 10 15

<210> 109

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 109

Phe Ala Thr Tyr Tyr Cys Met Gln Ser Tyr Glu Tyr Pro Phe Thr

1 5 10 15

<210> 110

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 110

Phe Ala Thr Tyr Tyr Ser Xaa Gln Ser Tyr Glu Tyr Pro Phe Thr

1 5 10 15

<210> 111

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 111

Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 10 15

<210> 112

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 112

Lys Arg Leu Ile Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Pro

1 5 10 15

<210> 113

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 113

Lys Arg Leu Ile Tyr Tyr Met Ser Ser Leu Ala Ser Gly Val Pro

1 5 10 15

<210> 114

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 114

Lys Arg Leu Ile Tyr Tyr Xaa Ser Ser Leu Ala Ser Gly Val Pro

1 5 10 15

<210> 115

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 115

Gln Gly Asp Ser Leu Arg Thr Tyr Tyr Ala Ser Trp Tyr Gln Gln

1 5 10 15

<210> 116

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 116

Val Leu Val Ile Tyr Gly Lys His Lys Arg Pro Ser Gly Ile Pro

1 5 10 15

<210> 117

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 117

Glu Ala Asp Tyr Tyr Cys Met Ser Arg Ser Ile Trp Gly Asn Pro

1 5 10 15

<210> 118

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 118

Glu Ala Asp Tyr Tyr Ser Xaa Ser Arg Ser Ile Trp Gly Asn Pro

1 5 10 15

<210> 119

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 119

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Thr

1 5 10 15

<210> 120

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 120

Gly Leu Glu Trp Ile Gly Ser Ile Ser His Thr Gly Asn Thr Tyr

1 5 10 15

<210> 121

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 121

Asn Pro Pro Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser

1 5 10 15

<210> 122

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 122

Asp Thr Ala Val Val Tyr Cys Ala Arg Gly Gly Gly Ile Ser Arg

1 5 10 15

<210> 123

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 123

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Phe

20 25 30

Asn Gly Asn Thr Tyr Leu Phe Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Arg Leu Ile Tyr Tyr Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Met Gln Ser

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 124

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 124

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 125

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 125

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro

20

<210> 126

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 126

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Ala Gly Ala Pro

20

<210> 127

<211> 98

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 127

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 128

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 128

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro

85 90 95

<210> 129

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 129

Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr

1 5 10

<210> 130

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 130

Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210> 131

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<400> 131

Trp Ile Asp Pro Asp Xaa Gly Asn Thr Ile Tyr Xaa Pro Lys Phe Gln

1 5 10 15

Gly

<210> 132

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 132

Arg Ser Thr Lys Ser Leu Xaa His Phe Asn Gly Asn Thr Tyr Leu Phe

1 5 10 15

<210> 133

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 133

Tyr Tyr Met Ser Xaa Leu Ala Ser

15

<210> 134

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<400> 134

Xaa Gln Ser Leu Glu Tyr Pro Phe Thr

15

<210> 135

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<400> 135

Trp Ile Asp Pro Asp Xaa Gly Asn

15

<210> 136

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<400> 136

Ser Thr Lys Ser Leu Xaa His Phe Asn Gly Asn Thr Tyr Leu

1 5 10

<210> 137

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 137

Tyr Tyr Met Ser Xaa

15

<210> 138

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 138

Arg Ser Thr Lys Ser Ile Leu His Phe Asn Gly Asn Thr Tyr Leu Phe

1 5 10 15

<210> 139

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 139

Ser Thr Lys Ser Ile Leu His Phe Asn Gly Asn Thr Tyr Leu

1 5 10

<210> 140

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 140

Arg Ser Thr Lys Ser Leu Leu His Phe Asn Gly Asn Ser Tyr Leu Phe

1 5 10 15

<210> 141

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 141

Ser Thr Lys Ser Leu Leu His Phe Asn Gly Asn Ser Tyr Leu

1 5 10

<210> 142

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 142

Tyr Ala Met Ser Asn Leu Ala Ser

15

<210> 143

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 143

Tyr Ala Met Ser Asn

15

<210> 144

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 144

Tyr Tyr Ala Ser Asn Leu Ala Ser

15

<210> 145

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 145

Tyr Tyr Ala Ser Asn

15

<210> 146

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 146

Tyr Tyr Met Ser Asn Leu Gln Ser

15

<210> 147

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 147

Met Gln Ser Tyr Glu Tyr Pro Phe Thr

15

<210> 148

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 148

Gln Ser Tyr Glu Tyr Pro Phe Thr

15

<210> 149

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 149

Met Gln Ser Leu Ser Tyr Pro Phe Thr

15

<210> 150

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 150

Gln Ser Leu Ser Tyr Pro Phe Thr

15

<210> 151

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 151

Met Gln Ser Leu Glu Thr Pro Phe Thr

15

<210> 152

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 152

Gln Ser Leu Glu Thr Pro Phe Thr

15

<210> 153

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 153

Met Gln Ser Leu Glu Tyr Pro Leu Thr

15

<210> 154

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 154

Gln Ser Leu Glu Tyr Pro Leu Thr

15

<210> 155

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 155

Met Gln Ser Leu Glu Tyr Pro Trp Thr

15

<210> 156

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 156

Gln Ser Leu Glu Tyr Pro Trp Thr

15

<210> 157

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 157

Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Gln Lys Phe Gln

1 5 10 15

Gly

<210> 158

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 158

Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Asp Ser Val Lys

1 5 10 15

Gly

<210> 159

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 159

Gly Phe Asn Ile Ala Asp Tyr Tyr Met Asn

1 5 10

<210> 160

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 160

Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Glu Pro Lys Phe Gln

1 5 10 15

Gly

<210> 161

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 161

Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 162

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 162

Trp Ile Asp Pro Asp Asn Gly Gln Thr Ile Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 163

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 163

Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Ala Lys Phe Gln

1 5 10 15

Gly

<210> 164

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 164

Trp Ile Asp Pro Asp Asn Gly Gln

15

<210> 165

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 165

Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Ala Phe Gln

1 5 10 15

Gly

<210> 166

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 166

Trp Ile Asp Pro Asp Gln Gly Asn Thr Ile Tyr Glu Pro Lys Val Gln

1 5 10 15

Gly

<210> 167

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (8)..(8)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (12)..(16)

<223> Xaa can be any naturally occurring amino acid

<400> 167

Trp Ile Asp Pro Asp Xaa Gly Xaa Thr Ile Tyr Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Gly

<210> 168

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(7)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (13)..(13)

<223> Xaa can be any naturally occurring amino acid

<400> 168

Arg Ser Thr Lys Ser Xaa Xaa His Phe Asn Gly Asn Xaa Tyr Leu Phe

1 5 10 15

<210> 169

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 169

Tyr Xaa Xaa Ser Xaa Leu Xaa Ser

15

<210> 170

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (4)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (8)..(8)

<223> Xaa can be any naturally occurring amino acid

<400> 170

Xaa Gln Ser Xaa Xaa Xaa Pro Xaa Thr

15

<210> 171

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (15)..(15)

<223> Xaa can be any naturally occurring amino acid

<400> 171

Trp Ile Asp Pro Asp Xaa Gly Asn Thr Ile Tyr Xaa Pro Lys Xaa Gln

1 5 10 15

Gly

<210> 172

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 172

Arg Ser Thr Lys Ser Leu Xaa His Phe Asn Gly Asn Thr Tyr Leu Phe

1 5 10 15

<210> 173

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 173

Tyr Tyr Xaa Ser Xaa Leu Ala Ser

15

<210> 174

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<400> 174

Xaa Gln Ser Xaa Glu Tyr Pro Phe Thr

15

<210> 175

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 175

Gly Phe Asn Ile Xaa Asp Tyr Tyr Met Asn

1 5 10

<210> 176

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (6)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (8)..(8)

<223> Xaa can be any naturally occurring amino acid

<400> 176

Trp Ile Asp Pro Asp Xaa Gly Xaa

15

<210> 177

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (5)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<400> 177

Ser Thr Lys Ser Xaa Xaa His Phe Asn Gly Asn Xaa Tyr Leu

1 5 10

<210> 178

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 178

Tyr Xaa Xaa Ser Xaa

15

<210> 179

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 179

Tyr Tyr Xaa Ser Xaa

15

<210> 180

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 180

Gln Ser Xaa Glu Tyr Pro Phe Thr

15

<210> 181

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 181

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 182

<211> 444

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 182

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Ser Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Leu Leu Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 183

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 183

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggcgt gcactccgag

60

gtgcagctgg tggaaagcgg aggaggcctg gtgcagcctg gaggaagcct gaggctgagc

120

tgtgccgcca gcggcttcaa catcaaggac tactacatga actgggtgag gcaggcccct

180

ggcaaaggac tggagtgggt gggctggatc gaccccgacc agggcaacac catctacgag

240

cccaagttcc agggcaggtt caccatcagc gccgacacca gcaagaacag cgcctacctg

300

cagatgaact ccctgagggc cgaggacacc gccgtgtact actgcgccag gaggctgctg

360

atggactact ggggccaggg cacactggtc accgtctcct cagcctccac caagggccca

420

tcggtcttcc ccctggcacc ctcctccaag agcacctctg ggggcacagc ggccctgggc

480

tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg

540

accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc

600

agcgtggtga ccgtgccctc cagcagcttg ggcacccaga cctacatctg caacgtgaat

660

cacaagccca gcaacaccaa ggtggacaag aaagttgagc ccaaatcttg tgacaaaact

720

cacacatgcc caccgtgccc agcacctgaa gccgctgggg caccgtcagt cttcctcttc

780

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg

840

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag

900

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc

960

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc

1020

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc

1080

cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc

1140

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtggggagagc

1200

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc

1260

ttcttcctct atagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc

1320

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg

1380

tcccccgga

1389

<210> 184

<211> 329

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 184

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 185

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 185

gacatccaga tgacccagtc cccttccagc ctgagcgctt ccgtgggcga cagggtgacc

60

atcacctgca ggtccaccaa gtccctgtcc cacttcaacg gcaacaccta cctgttctgg

120

taccagcaga agcccggcaa ggcccccaag aggctgatct actacatgtc ctccctggcc

180

tccggagtgc cctccaggtt ctccggatcc ggctccggca ccgacttcac cctgaccatc

240

tcctccctgc agcccgagga tttcgccacc tactactgcc agcagtccct ggagtacccc

300

ttcaccttcg gcggcggcac caaggtggag atcaaacgaa ctgtggctgc accatctgtc

360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg

420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa

480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc

540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa

600

gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt

657

<210> 186

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 186

gacatccaga tgacccagtc cccttccagc ctgagcgctt ccgtgggcga cagggtgacc

60

atcacctgca ggtccaccaa gtccctgtcc cacttcaacg gcaacaccta cctgttctgg

120

taccagcaga agcccggcaa ggcccccaag aggctgatct actacatgtc ctccctggcc

180

tccggagtgc cctccaggtt ctccggatcc ggctccggca ccgacttcac cctgaccatc

240

tcctccctgc agcccgagga tttcgccacc tactactgcc agcagtccct ggagtacccc

300

ttcaccttcg gcggcggcac caaggtggag atcaaa

336

<210> 187

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 187

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggcgt gcactcc

57

<210> 188

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 188

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 189

<211> 1335

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 189

gaggtgcagc tggtggaaag cggaggaggc ctggtgcagc ctggaggaag cctgaggctg

60

agctgtgccg ccagcggctt caacatcaag gactactaca tgaactgggt gaggcaggcc

120

cctggcaaag gactggagtg ggtgggctgg atcgaccccg accagggcaa caccatctac

180

gagcccaagt tccagggcag gttcaccatc agcgccgaca ccagcaagaa cagcgcctac

240

ctgcagatga actccctgag ggccgaggac accgccgtgt actactgcgc caggaggctg

300

ctgatggact actggggcca gggcacactg gtcaccgtct cctcagcctc caccaagggc

360

ccatcggtct tccccctggc accctcctcc aagagcacct ctggggggcac agcggccctg

420

ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc

480

ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc

540

agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg

600

aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa

660

actcacacat gcccaccgtg cccagcacct gaagccgctg gggcaccgtc agtcttcctc

720

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg

780

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg

840

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg

900

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag

960

gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag

1020

ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag

1080

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtggggag

1140

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc

1200

tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc

1260

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc

1320

ctgtcccccg gaaaa

1335

<210> 190

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 190

gaggtgcagc tggtggaaag cggaggaggc ctggtgcagc ctggaggaag cctgaggctg

60

agctgtgccg ccagcggctt caacatcaag gactactaca tgaactgggt gaggcaggcc

120

cctggcaaag gactggagtg ggtgggctgg atcgaccccg accagggcaa caccatctac

180

gagcccaagt tccagggcag gttcaccatc agcgccgaca ccagcaagaa cagcgcctac

240

ctgcagatga actccctgag ggccgaggac accgccgtgt actactgcgc caggaggctg

300

ctgatggact actggggcca gggcacactg gtcaccgtct cctca

345

<210> 191

<211> 1332

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 191

gaggtgcagc tggtggaaag cggaggaggc ctggtgcagc ctggaggaag cctgaggctg

60

agctgtgccg ccagcggctt caacatcaag gactactaca tgaactgggt gaggcaggcc

120

cctggcaaag gactggagtg ggtgggctgg atcgaccccg accagggcaa caccatctac

180

gagcccaagt tccagggcag gttcaccatc agcgccgaca ccagcaagaa cagcgcctac

240

ctgcagatga actccctgag ggccgaggac accgccgtgt actactgcgc caggaggctg

300

ctgatggact actggggcca gggcacactg gtcaccgtct cctcagcctc caccaagggc

360

ccatcggtct tccccctggc accctcctcc aagagcacct ctggggggcac agcggccctg

420

ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc

480

ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc

540

agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg

600

aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa

660

actcacacat gcccaccgtg cccagcacct gaagccgctg gggcaccgtc agtcttcctc

720

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg

780

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg

840

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg

900

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag

960

gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag

1020

ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag

1080

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtggggag

1140

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc

1200

tccttcttcc tctatagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc

1260

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc

1320

ctgtcccccg ga

1332

<210> 192

<211> 987

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 192

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg

60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg

120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca

180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc

240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc

300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgctggggca

360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc

660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag

720

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc

780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg

840

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg

900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

960

cagaagagcc tctccctgtc ccccgga

987

<210> 193

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 193

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg

60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg

120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca

180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc

240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc

300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgctggggca

360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc

660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag

720

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc

780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg

840

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg

900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

960

cagaagagcc tctccctgtc ccccggaaaa

990

<210> 194

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 194

cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct

60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag

120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac

180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag

240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag

300

agcttcaaca ggggagagtg t

321

<210> 195

<211> 98

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 195

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 196

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 196

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro

85 90 95

<210> 197

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_sign

<222> (3)..(5)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_sign

<222> (7)..(7)

<223> Xaa can be any naturally occurring amino acid

<400> 197

Gln Ser Xaa Xaa Xaa Pro Xaa Thr

15

<---

Claims (73)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin, where the antibody or fragment is at least one antibody or fragment selected from the group consisting of: (a) an antibody or antigen binding fragment thereof comprising a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 11; CDR-L2 containing the amino acid sequence SEQ ID NO: 12; CDR-L3 containing the amino acid sequence SEQ ID NO: 13; Heavy chain CDR1 (CDR-H1) containing the amino acid sequence of SEQ ID NO: 8; CDR-H2 containing the amino acid sequence SEQ ID NO: 9; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 10; (b) an antibody or antigen binding fragment thereof comprising a CDR-L1 containing the amino acid sequence of SEQ ID NO: 17; CDR-L2 containing the amino acid sequence SEQ ID NO: 18; CDR-L3 containing the amino acid sequence SEQ ID NO: 19; CDR-H1 containing the amino acid sequence SEQ ID NO: 14; CDR-H2 containing the amino acid sequence SEQ ID NO: 15; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 16; (c) an antibody or antigen binding fragment thereof comprising a VL region containing the amino acid sequence of SEQ ID NO: 7, and a VH region containing the amino acid sequence of SEQ ID NO: 6; (d) an antibody or antigen binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-66 and a VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 34- 38; (e) an antibody or antigen binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47 and 92 and a VH region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 39 and 88-91; (f) an antibody or antigen binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 67-69 and a VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 93; (g) an antibody or antigen binding fragment thereof comprising a VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 47-69 and 92 and a VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 34-46, 88-91 and 93; (h) an antibody or antigen binding fragment thereof comprising a light chain (LC) region containing the amino acid sequence of SEQ ID NO: 5 and a heavy chain (HC) region containing the amino acid sequence of SEQ ID NO: 2; (i) an antibody or antigen binding fragment thereof comprising an LC region containing the amino acid sequence of SEQ ID NO: 5 and an HC region containing the amino acid sequence of SEQ ID NO: 3; (j) an antibody or antigen binding fragment thereof comprising an LC region containing the amino acid sequence of SEQ ID NO: 123 and an HC region containing the amino acid sequence of SEQ ID NO: 124 or 182; (k) an antibody or antigen binding fragment thereof comprising a VL region encoded by the nucleic acid sequence SEQ ID NO: 186 and a VH region encoded by the nucleic acid sequence SEQ ID NO: 190; And (l) an antibody or antigen binding fragment thereof comprising an LC region encoded by the nucleic acid sequence SEQ ID NO: 185 and an HC region encoded by the nucleic acid sequence SEQ ID NO: 189 or 191. 2. An isolated antibody or an antigen-binding fragment thereof according to claim 1, where the antibody or an antigen-binding fragment thereof contains: light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 11; CDR-L2 containing the amino acid sequence SEQ ID NO: 12; CDR-L3 containing the amino acid sequence SEQ ID NO: 13; Heavy chain CDR1 (CDR-H1) containing the amino acid sequence of SEQ ID NO: 8; CDR-H2 containing the amino acid sequence SEQ ID NO: 9; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 10. 3. An isolated antibody or an antigen-binding fragment thereof according to claim 1, where the antibody or an antigen-binding fragment thereof contains: CDR-L1 containing the amino acid sequence SEQ ID NO: 17; CDR-L2 containing the amino acid sequence SEQ ID NO: 18; CDR-L3 containing the amino acid sequence SEQ ID NO: 19; CDR-H1 containing the amino acid sequence SEQ ID NO: 14; CDR-H2 containing the amino acid sequence SEQ ID NO: 15; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 16. 4. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising a VL region containing an amino acid sequence at least 95% identical to SEQ ID NO: 7 and a VH region containing an amino acid sequence at least 95% identical to SEQ ID NO: 6 . 5. An isolated antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-4, containing a VL region containing the amino acid sequence of SEQ ID NO: 7, and a VH region containing the amino acid sequence of SEQ ID NO: 6. 6. An isolated antibody or an antigen-binding fragment thereof according to any one of claims. 1-5, containing an LC region containing an amino acid sequence at least 95% identical to SEQ ID NO: 5, and an HC region containing an amino acid sequence at least 95% identical to SEQ ID NO: 2 or 3. 7. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 6, comprising an LC region containing the amino acid sequence of SEQ ID NO: 5, and an HC region containing the amino acid sequence of SEQ ID NO: 2 or 3. 8. An isolated antibody or antigen-binding fragment thereof that specifically binds the αvβ8 integrin, wherein the antibody or antigen-binding fragment comprises a VH region containing an amino acid sequence of any of SEQ ID NOs: 6, 34-46, 88-91 and 93, and/or VL a region containing the amino acid sequence of any of SEQ ID NOs: 7, 47-69 and 92. 9. An isolated antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin, where the antibody or antigen-binding fragment contains: (i) an HC antibody containing the amino acid sequence of SEQ ID NO: 2 or 3; And (ii) An antibody LC containing the amino acid sequence of SEQ ID NO: 5. 10. An isolated antibody that specifically binds αvβ8 integrin, comprising an LC consisting of the amino acid sequence of SEQ ID NO: 5, and an HC consisting of the amino acid sequence of SEQ ID NO: 2 or 3. 11. An isolated antibody that specifically binds αvβ8 integrin, containing: a VL region of an antibody containing CDR-L1, CDR-L2 and CDR-L3 from the VL region containing the amino acid sequence of SEQ ID NO: 7; And VH region of an antibody containing CDR-H1, CDR-H2 and CDR-H3 from the VH region containing the amino acid sequence SED ID NO: 6. 12. The isolated antibody of claim 11, comprising an antibody heavy chain constant region containing the amino acid sequence of SEQ ID NO: 181 or 184, and an antibody light chain constant region containing the amino acid sequence of SEQ ID NO: 83. 13. An isolated antibody that specifically binds αvβ8 integrin, containing: a) a VL region of an antibody containing the first, second and third CDRs of the VL region containing the amino acid sequence of SEQ ID NO: 7; a VH region of an antibody comprising the first, second and third CDRs of the VH region containing the amino acid sequence of SEQ ID NO: 6; an antibody light chain constant region (CL) containing the amino acid sequence of SEQ ID NO: 83; And an antibody heavy chain constant region (CH) comprising the amino acid sequence of SEQ ID NO: 181 or 184; b) a VL region of an antibody containing an amino acid sequence at least 95% identical to SEQ ID NO: 7; and a VH region of an antibody containing an amino acid sequence at least 95% identical to SEQ ID NO: 6; or c) an antibody LC region containing an amino acid sequence at least 95% identical to SEQ ID NO: 5, and an antibody HC region containing an amino acid sequence at least 95% identical to SEQ ID NO: 2 or 3. 14. An isolated antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-13, containing a human IgG1 Fc region containing one or more substitutions selected from positions L234, L235 and G237, numbered according to the Eu numbering according to Kabat. 15. The isolated antibody or antigen binding fragment thereof according to claim 14, wherein the human IgG1 Fc region comprises one or more of L234A, L235A and G237A. 16. An isolated antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-15, where the antibody is a humanized antibody, a human antibody, a mouse antibody, a chimeric antibody, or a camel antibody. 17. An isolated antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-13 or 16, wherein the antibody heavy chain isotype is selected from IgG1, IgG2, IgG3, IgG4, or any variant thereof; and/or wherein the light chain constant region is selected from kappa or lambda. 18. The isolated antibody or antigen-binding fragment thereof according to claim 17, wherein the isotype of the heavy chain of the antibody is IgG1, and/or wherein the light chain constant region is a kappa light chain. 19. A pharmaceutical composition for the treatment or prevention of cancer or a condition mediated by the activity of αvβ8 integrin, containing an antibody or an antigen-binding fragment according to any one of paragraphs. 1-18 and a pharmaceutically acceptable carrier or excipient. 20. The pharmaceutical composition according to claim 19, containing i) an antibody or an antigen-binding fragment thereof containing an antibody heavy chain encoded by the amino acid sequence SEQ ID NO: 2 and an antibody light chain encoded by the amino acid sequence SEQ ID NO: 5, ii) an antibody or an antigen binding fragment thereof comprising an antibody heavy chain encoded by the amino acid sequence of SEQ ID NO: 3 and an antibody light chain encoded by the amino acid sequence of SEQ ID NO: 5 or iii) both. 21. An isolated nucleic acid that encodes the amino acid sequences of an antibody or an antigen-binding fragment thereof according to any one of claims. 1-18. 22. The isolated nucleic acid according to claim 21, where the isolated nucleic acid encodes a VH region, a VL region or both of an antibody or an antigen-binding fragment thereof, and wherein said nucleic acid contains: nucleic acid sequence SEQ ID NO: 190, nucleic acid sequence SEQ ID NO: : 186 or both. 23. The isolated nucleic acid according to claim 21, where the isolated nucleic acid encodes a heavy chain constant region, a light chain constant region, or both of an antibody or an antigen binding fragment thereof, and where the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 192 or 193; nucleic acid sequence SEQ ID NO: 194; or both. 24. The isolated nucleic acid according to claim 21, where the isolated nucleic acid encodes HC, LC or both antibodies or an antigen-binding fragment thereof, and where the specified nucleic acid contains: the nucleic acid sequence SEQ ID NO: 189 or 190; nucleic acid sequence SEQ ID NO 185; or both. 25. The isolated nucleic acid according to claim 21, where the isolated nucleic acid contains at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100 of the nucleic acid sequence % identical to SEQ ID NO: 189 or SEQ ID NO: 191; a nucleic acid sequence at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98% or 100% identical to SEQ ID NO: 185; or both. 26. An expression vector containing a nucleic acid according to any one of paragraphs. 21-25. 27. A host cell for obtaining an isolated antibody or an antigen-binding fragment thereof according to any one of claims. 1-18, containing the isolated nucleic acid according to any one of paragraphs. 21-25 or the expression vector according to claim 26. 28. The host cell of claim 27, wherein the host cell is a mammalian cell selected from the group consisting of CHO cells, COS cells, HEK-293 cells, NS0 cells, PER.C6® cells and Sp2.0 cells. 29. A method for producing an isolated antibody or an antigen-binding fragment thereof according to any one of claims. 1-18, comprising culturing the host cell of claim 27 under conditions where the antibody or fragment is expressed by the host cell, and isolating the antibody or fragment. 30. A method of reducing αvβ8 integrin activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims. 1-18 or the pharmaceutical composition according to claim 19 or 20. 31. A method of treating or preventing a cancer or condition mediated by αvβ8 integrin activity, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims. 1-18 or the pharmaceutical composition according to claim 19 or 20. 32. The method of claim 31, comprising further administering to the subject a cytotoxic agent, a cytotoxic agent, a chemotherapeutic agent, hormonal therapy, a vaccine, immunotherapy, surgery, radiation, cryosurgery, chemotherapy, or a combination thereof. 33. The method of claim 32, wherein the additional administration is simultaneous, sequential or separate from the administration of a therapeutically effective amount of the antibody or antigen binding fragment or pharmaceutical combination. 34. The method of claim 32, wherein the immunotherapy comprises an immune checkpoint molecule modulator selected from the group consisting of anti-PD1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, soluble CTLA-4 fusion protein and combinations thereof, and where the anti-PD-L1 antibody is not avelumab. 35. Method according to any one of paragraphs. 31-34, wherein the cancer is selected from the group consisting of squamous cell carcinoma of the head and neck, clear cell or papillary cell type renal cell carcinoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, squamous cell lung cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, uterine cancer, melanoma, urothelial carcinoma and combinations thereof. 36. A method for determining αvβ8 integrin in a sample, tissue or cell using an antibody or an antigen-binding fragment thereof according to claims. 1-18, comprising contacting a sample, tissue or cell with an antibody or an antigen-binding fragment thereof and detecting an αvβ8 integrin in the sample, tissue or cell by detecting the antibody or an antigen-binding fragment thereof. 37. A kit for the treatment or prevention of cancer or condition mediated by αvβ8 integrin activity, comprising an antibody or fragment according to any one of claims. 1-18 or the pharmaceutical composition according to claim 19 or 20. 38. The kit of claim 37, further comprising an immune checkpoint molecule modulator selected from the group consisting of anti-PD1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, soluble CTLA -4 fusion proteins and combinations thereof, and where the anti-PD-L1 antibody is not avelumab. 39. An antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-18 or the pharmaceutical composition according to claim 19 or 20 for use in reducing αvβ8 integrin activity in a subject in need thereof for the treatment of cancer mediated by αvβ8 integrin activity. 40. An antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-18 or a pharmaceutical composition according to claim 19 or 20 for use in the treatment of cancer mediated by αvβ8 integrin activity. 41. An antibody or antigen-binding fragment thereof or a pharmaceutical composition for use according to claim 40, wherein the antibody or antigen-binding fragment or pharmaceutical composition thereof is intended to be administered simultaneously, sequentially or separately in combination with immunotherapy. 42. An antibody or antigen-binding fragment thereof or a pharmaceutical composition for use according to claim 41, where the combination provides a synergistic therapeutic effect. 43. An antibody or antigen-binding fragment thereof or a pharmaceutical composition for use according to claim 42, wherein the cancer is selected from the group consisting of squamous cell carcinoma of the head and neck, clear cell or papillary cell type renal cell carcinoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, squamous cell lung cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, uterine cancer, melanoma, urothelial carcinoma and combinations thereof, optionally, wherein the antibody or antigen-binding fragment thereof or pharmaceutical composition or combination is intended for use in conjunction with the administration of immunotherapy or radiation therapy. 44. The use of an antibody or an antigen-binding fragment according to any one of paragraphs. 1-18 or a pharmaceutical composition according to claim 19 or 20 for the treatment of cancer mediated by αvβ8 integrin activity. 45. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18 in the manufacture of a medicament for the treatment of cancer mediated by αvβ8 integrin activity. 46. A method of treating cancer mediated by αvβ8 integrin activity, comprising administering to a subject in need thereof a therapeutically effective amount of (i) an antibody or antigen-binding fragment thereof that specifically binds αvβ8 integrin, and (ii) an anti-PD1 modulator, anti-PD- L1 or anti-PD-L2 immune checkpoint molecules. 47. The method of claim 46, wherein the cancer is squamous cell carcinoma. 48. The method of claim 46, wherein the cancer is breast cancer or colon cancer. 49. The method of claim 46, wherein the modulator is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, and an anti-PD-L2 antibody.