NZ768752A

NZ768752A - Anti-human 4-1 bb antibodies and use thereof

Info

Publication number: NZ768752A
Application number: NZNZ768752A
Authority: NZ
Inventors: Young Ho Kim; Byoung S Kwon; Joong Won Lee; Seoung Joo Lee; Ho Sik Oh
Original assignee: Eutilex Co Ltd
Filing date: 2020-10-08
Publication date: 2020-10-30

Abstract

Provided are anti-human 4-1BB antibodies and fragments thereof with the specific mutations at position 94W of the light chain, and positions 5V, 68V, 91T, and 101K of the heavy chain that are not found in a reference anti-human 4- 1BB antibody, where said features may improve certain characteristics of the antibody relative to a reference antibody. Various in vitro and in vivo methods and reagents related to anti-human 4-1BB antibodies described herein are also provided. Methods include, for example, inducing T-cell proliferation, inducing T cell secretion of IFNγ, as well as detection, prevention, and/or therapeutic treatment of cancer using an anti-human 4-1BB antibody or fragment thereof. of the antibody relative to a reference antibody. Various in vitro and in vivo methods and reagents related to anti-human 4-1BB antibodies described herein are also provided. Methods include, for example, inducing T-cell proliferation, inducing T cell secretion of IFNγ, as well as detection, prevention, and/or therapeutic treatment of cancer using an anti-human 4-1BB antibody or fragment thereof.

Description

UMAN 4-IBB ANTIBODIES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the beneﬁt of US Patent Application No. 62/443,281, ﬁled on January 06, 2017, the disclosure of which is incorporated herein by reference in its ty.

BACKGROUND Cancer remains one of the leading causes of death in the world. Recent statistics report that 13% of the world population dies from cancer. ing to estimates from the lntemational Agency for Research on Cancer (IARC), in 2012 there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide. By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths due to population growth and aging and re to risk factors such as smoking, unhealthy diet and al inactivity. Further, pain and medical expenses for cancer treatment cause reduced y of life for both cancer patients and their families. It is apparent that, above all, cancer is a disease for which it is necessary to urgently find improved treatment s.

The present disclosure provides, among other things, antibodies and fragments thereof that bind to a human 4-lBB polypeptide. In some aspects, provided anti-human 4- 188 antibodies and fragments thereof are variants of a nce anti-human 4-lBB antibody in that they contain one or more panicular structural features that are not found in the reference anti- human 4-lBB antibody. The present disclosure encompasses a recognition that provided variant anti-human 4—l BB antibodies have improved properties relative to a reference anti-human 4-1 BB antibody lacking one or more structural features described . In some embodiments, ed anti-human 4-lBB antibodies and nts thereof have one or more improved properties, such as, for example, improved binding affinity, improved induction of T cell proliferation (e.g., proliferation of CD8' T cells), increased ability to induce IFNy production by T cells (e.g., proliferation of CD8‘ T cells), improved ability to reduce and/or eliminate cancer proliferation in viva (e.g., at a lower dose).

In some embodiments, an antiIBB antibody or antigen-binding antibody fragment includes 1, 2, or 3 heavy chain CDR sequences that are or include a sequence of SEQ ID NOs: 5 to 8. In some embodiments, an antiIBB antibody or antigen-binding antibody fragment includes one or more of: a heavy chain CDRI that is or es a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8. In some embodiments, an anti IBB antibody or antigen-binding antibody fragment includes each of: a heavy chain CDRI that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8.

In some embodiments, an anti1BB dy or antigen-binding antibody fragment includes 1, 2, or 3 light chain CDR sequences that are or include a sequence of SEQ ID NOS: 1-4. In some embodiments, an anti-4—IBB antibody or antigen-binding dy fragment es one or more of: a light chain CDRI that is or includes a sequence of SEQ ID NO: I, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4. In some embodiments, an antiI BB antibody or antigen-binding antibody fragment includes each of: a light chain CDRI that is or includes a sequence of SEQ ID NO: I, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4.

In some embodiments, an anti1BB antibody or n-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDRI that is or es a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a ce of SEQ ID NO: 7 or 8, and/or a light chain variable domain that includes a light chain CDRI that is or includes a sequence of SEQ ID NO: I, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 4.

In some embodiments, an antiIBB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework I (FRI) region comprising a sequence of SEQ ID NO' 16 or 17. In some ments, an -IBB dy or n—binding antibody fragment es a heavy chain variable domain that includes a heavy chain framework 3 (FR3) region sing a sequence of any one of SEQ ID NOS: 18-20. In some embodiments, an anti—44 BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain Framework 1 (FRI) region comprising a sequence of SEQ ID NO: 16 or 17 and a heavy chain framework 3 (FR3) region comprising a sequence of any one of SEQ [D NOs: 18-20.

In some embodiments, an anti1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or es a sequence selected from SEQ ID NOS: 1 1—14.

In some embodiments, an -lBB antibody or antigen-binding antibody nt es a heavy chain variable domain that is or includes a ce at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID NOS: 1 1-14. In some embodiments, an antiIBB antibody or antigen- binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID N05: 11-14.

In some embodiments, an antiIBB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody nt that includes a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10. In some embodiments, an antil BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 9959/6 cal to a sequence of SEQ ID NO: 9 or 10. In some embodiments, an anti1BB antibody or n-binding antibody nt es a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10.

In some embodiments, an anti—4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an dy or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOS: 1 1-14 and a light chain variable domain that is or includes a sequence of SEQ ID NO: 10. In sotne embodiments, an anti1 BB antibody or n-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 959/ , 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID N05: 1 1-14 and a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence of SEQ ID NO: 10. In some embodiments, an anti—4- I BB antibody or antigen-binding antibody fragment includes a heavy chain le domain that is or includes a sequence selected from SEQ ID NOS: 1 1-14 and a light chain variable domain that is or es a sequence of SEQ ID NO: 10.

In some embodiments, a provided anti—human 4-lBB antibody or fragment thereof is an agonistic antibody. In some embodiments, a provided anti—human 4-lBB antibody or fragment thereof is terized as having superior agonistic activity than a humanized anti- human 4-lBB antibody 94G] (i.e., an antibody ing light chain and heavy chain variable domains of SEQ ID NOS: 9 and l 1, respectively). In some ments, a ed anti-human 4-1BB antibody or fragment thereof is characterized as having improved binding affinity than a humanized anti-human 4-1BB antibody 94Gl (i.e., an antibody including light chain and heavy chain variable domains of SEQ ID NOS: 9 and l 1, respectively).

In some embodiments, a provided anti-human 4-IBB antibody or fragment thereof is or ses a humanized antibody. In some embodiments, a provided anti-human 4- 188 antibody or fragment thereof includes a human globulin constant domain, wherein the constant domain is selected from an IgGl or a variant thereof, an IgGZ or a variant thereof, an lgG4 or a variant thereof, an lgA or a variant thereof, an IgE or a variant thereof, an IgM or a variant f, and an IgD or a variant thereof. In some ments, a provided uman 4- 188 antibody or fragment thereof is or comprises a human IgG1. In some embodiments, an IgGl is or comprises a sequence that is at least 95% identical to SEQ ID NO: 22 or 23, In some embodiments, a provided anti-human 4—lBB antibody or fragment thereof is a monoclonal antibody.

In some embodiments, a provided anti-human 4-lBB antibody or fragment thereof is a full length antibody. In some ments, a provided anti-human 4-lBB antibody or fragment thereof is a Fab fragment, 3 Fab' fragment, a F(ab')2 fragment, a Fv fragment, a ide-bonded Fv fragment, a scFv fragment, a single domain antibody, humabody, nanobody, or a diabody.

In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof has a binding affinity (KD) for a human 4-1BB molecule of I X 10‘7 to 1X 10'l2 M. In some embodiments, a provided anti-human 4-lBB antibody or fragment f has a binding affinity (KD) for a human 4-lBB molecule of l x 10'8 to l>< IO'U M. In some embodiments, a provided anti-human 4-IBB antibody or fragment thereof has a binding afﬁnity (K0) for a human 4-lBB molecule of l X l 0'9 to IX 10'12 M. In some embodiments, a provided anti-human 4-IBB antibody or fragment thereof has a binding affinity (KD) for a human 4-lBB molecule of l x10'l0 to IX 10' In some embodiments, a provided anti-human 4-IBB antibody or fragment thereof binds to an epitope within the extracellular domain of a human 4- l BB polypeptide. In some embodiments, a ed anti-human 4-lBB antibody or fragment thereof binds to an epitope within the extracellular domain of human 4-IBB. In some embodiments, binding of a provided anti-human 4- [BB antibody or fragment thereof is abrogated by one or more mutations at positions N30, D38, N39, and R4] of SEQ ID NO: 44.

In some embodiments, a provided anti-human 4-IBB antibody or fragment thereof fails to bind or weakly binds a non-primate 4- [BB polypeptide. In some embodiments, a provided anti-human 4-IBB antibody or fragment f fails to bind or weakly binds a canine 4-IBB polypeptide.

In some ments, the present disclosure provides c acid molecules encoding an antil BB dy or antigen-binding fragment. In some embodiments, the present disclosure provides vectors that include a c acid molecule encoding an anti I BB antibody or antigen-binding fragment. In some embodiments, the present disclosure provides host cells that include a vector and/or nucleic acid molecule encoding an antil BB antibody or antigen-binding fragment. In some embodiments, a host cell is selected from a ial, yeast, insect or mammalian cell. In some embodiments, a is selected from the group consisting of lion/i, P.pas!0ris, Sf‘9, COS, HEK293, CHO and a mammalian lymphocyte.

In some embodiments, the present disclosure provides ceutical itions that include an anti—4-l BB antibody or antigen-binding fragment and a phannaceutically acceptable carrier. In some embodiments, the present sure provides pharmaceutical compositions that include a nucleic acid and/or vector encoding an antiIBB antibody or antigen-binding nt and a ceutically able carrier.

In some embodiments, the present disclosure provides methods oftreating a subject in need thereof, the method sing administering to the subject a composition that comprises or delivers an antiIBB antibody or antigen-binding fragment. In some embodiments, the present disclosure provides methods of treating a subject in need thereof, the method comprising administering to the subject a composition that comprises or rs a nucleic acid and/or vector encoding an antilBB antibody or antigen-binding fragment. In some embodiments, a subject has or is at risk for developing cancer. [0021'] In some embodiments, the t disclosure provides methods of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a composition that comprises or delivers an antil BB antibody or antigen-binding fragment. In some embodiments, the present disclosure provides methods of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a ition that comprises or delivers a c acid and/or vector encoding an anti IBB antibody or n-binding fragment. In some embodiments, a subject has or is at risk for ping cancer.

In some embodiments, the present disclosure provides methods of enhancing an immune response in a t in need thereof, the method comprising administering to the subject a composition that comprises or delivers an anti l BB antibody or antigen—bindi ng fragment. In some embodiments, the present disclosure provides methods of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a composition that comprises or delivers a nucleic acid and/or vector encoding an anti IBB antibody or antigen-binding fragment. In some embodiments, a subject has or is at risk for developing cancer.

In some embodiments, a cancer to be treated by a method of the present disclosure in a t is selected from a bladder cancer, breast cancer, al cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder , intestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung , ma, melanoma, mesothelioma, n cancer, primary neal cancer, salivary gland cancer, sarcoma, h cancer, thyroid cancer, pancreatic cancer, and prostate cancer.

In some ments, a composition comprises or delivers an anti-human 4-lBB antibody of the present disclosure or an antigen-binding fragment thereof at a dose of 0.0] mg/kg to ICC mg/kg. In some embodiments, a composition comprises or delivers an anti-human 4-IBB antibody or an antigen-binding fragment f at a dose of about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 01 mg/kg, 0.25 mg/kg, 05 mg/kg, 0.75 mg/kg, l mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.

In some embodiments, anti-human 4-lBB antibodies and/or fragments thereof and/or compositions comprising the same are characterized by inducing increased T cell proliferation (e.g., CD8' T cell proliferation) and/or increased IFNY secretion by T cells (e.g., CD8' T cells) in a subject.

In some embodiments, the present disclosure provides methods that include administering to the t a composition that comprises or delivers an antil BB antibody or antigen-binding fragment to a t that has been administered or will be administered one or more additional anticancer therapies. In some ments, the t disclosure es s that include administering to the subject a composition that comprises or delivers an -l BB antibody or antigen-binding fragment to a subject that has been administered or will be administered one or more of ionizing ion, a chemotherapeutic agent, an antibody agent, and a ased therapy, such that the subject receives treatment with both.

In some embodiments, the present disclosure provides methods that include administering to the subject a composition that comprises or delivers an antilBB antibody or n-binding fragment to a subject that has been administered or will be administered one or more of an immune checkpoint tor, IL-12, GM-CSF, an anti-CD4 agent, fluorouracil, doxombicin, irinotecan, paclitaxel, cisplatin, or cyclophosphamidei In some embodiments, the present disclosure provides methods that include administering to the subject a composition that comprises or delivers an anti1BB antibody or antigen-binding fragment to a subject that has been administered or will be administered a composition comprising an immune checkpoint inhibitor, such that the subject receives treatment with both. In some embodiments, an immune checkpoint inhibitor is an agent that ts PD-l signaling. In some embodiments, an agent that inhibits PD—l signaling is an anti-PD-l antibody.

In some embodiments, an anti-PD-l antibody is nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.

In some embodiments, the present disclosure provides methods of determining a dose of an anti—4-l BB antibody or antigen binding fragment f for therapeutic treatment of a subject in need thereof. In some ments, such a method includes (i) providing or obtaining a measurement of secreted IFN-gamma in a biological sample from the subject, wherein the subject has been administered a composition that comprises or delivers an amount of an anti-4— IBB antibody or antigen-binding nt bed ; and (ii) comparing the measurement of ed IFN-gamma to a nce value, where if the measurement of secreted IFN-gamma is higher or lower than the reference value, adjusting the amount of an -lBB antibody or antigen binding fragment thereof to be administered, thereby determining a dose for therapeutic treatment of a subject, In some ments, a reference value comprises an index value which includes a value d from one or more healthy subjects, a value derived from one or more cancer diagnosed subject or a value derived from a cancer risk prediction algorithm.

In some embodiments, a biological sample is a sample of whole blood, plasma, or serum. In some embodiments, a subject has or is at risk for developing cancer. In some embodiments, a cancer is selected from a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer. intestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal , salivary gland cancer, sarcoma, h , thyroid cancer, pancreatic cancer, and prostate cancer.

In some embodiments, the present disclosure provides methods for increasing secretion of IFN-y by a cell in vivo or in vitro that include: contacting the cell with an anti 1 BB antibody or antigen-binding fragment described herein.

] In some embodiments, the present disclosure provides methods ex vivo proliferation or isolation of activated T cells that include: ting a population of T cells with an antiIBB antibody or antigen—binding fragment described herein, thereby increasing proliferation of activated T cells.

In some embodiments, the present disclosure provides methods for isolating antigen-specific activated T cells that include one or more steps of: (a) culturing eral blood mononuclear cells (PBMC) in a medium together with a peptide of an epitope of interest and IL- 2; (b) inducing 4-] BB expression in the cultured cells by adding the peptide of the epitope of interest; (c) contacting the cultured cells with a surface coated with an anti—4—IBB antibody or antigen—binding fragment described herein, wherein cultured cells expressing 4-1BB adhere to WO 27787 PCT/lBZOIS/(NMI043 the coated surface; and (d) removing unattached cells, thereby isolating antigen—speciﬁc activated T cells. In some embodiments, activated T cells are CD8’ T cells.

In some embodiments, the present disclosure provides methods for treating or preventing cancer in a subject in need thereof that includes stering to the subject a composition that includes a therapeutically effective amount of activated T cells produced by any of the method described herein. In some embodiments, a cancer is selected from a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal , head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and te cancer. In some embodiments, a composition es least 109, at least 10'" cells, or more than 10'0 activated T cells. In some embodiments. activated T cells are CD8‘ T cells.

Also provided, among other things, are technologies for characterizing anti- human 4-IBB antibodies and/or fragments thereof as described herein and/or compositions comprising the same. In some embodiments, provided are methods for characterizing anti- human 4- 1 BB antibodies and/or nts thereof and/or compositions comprising the same binding to AML cells (e.g., HL60). In some embodiments, provided are methods for characterizing anti-human 4-lBB antibodies and/or nts thereof and/or compositions comprising the same are by ELISA, immunohistochemistry, Biacore g assays, mass spectrometry, isoelectric focusing (IEF) tography, and/or western blot.

The present disclosure provides s technologies related to making or manufacturing uman 4-1BB antibodies and/or fragments thereof as bed herein and/or compositions containing said antibodies or fragments thereof.

As used in this application, the terms “about” and “approximately” are used as equivalents. Any citations to publications, patents, or patent applications herein are incorporated by reference in their entirety. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not tion. Various s and modiﬁcations within the scope of the ion will become apparent to those skilled in the art from the detailed ption.

BRIEF DESCRIPTION OF THE DRAWING The g ed , which is comprised of the following Figures, is for illustration purposes only and not for limitation. The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conjunction with the accompanying figures in which: depicts human 4-1BB extracellular domain (ECD) constructs. At the top is a schematic of a full length 4-IBB ECD (167 amino acids), and below is shown various fragments ofa 4-lBB ECD: RI (1-55 aa), R2 (56-1 l0 aa), R3 (1 10-167 aa), Rl.l (1-45 aa), R12 (I-35 aa), R13 (1 1-55 aa), R1.4 (21-55 aa) Rl.5 (I-25 aa) and Rl.6 (1-30 aa). Each of these 4-IBB ECD constructs were fused with G‘ST. FIG. IB depicts a western blot showing binding of an exemplary humanized antiIBB antibody to 4-1BB ECD fusion constructs as described in . As shown, an ary humanized antiIBB antibody binds to a full length 4-IBB ECD fusion polypeptide and to the RI fusion polypeptide, but not to the R2 or R3 fusion polypeptides. Molecular size markers are indicated in kDa on the left. depicts an GE gel of whole cell extracts from cells expressing 4-IBB ECD fusion constructs. Fusion constructs as described above in were expressed in E. coli BL2| cells induced with 1 mM IPTG, and whole cell extracts resolved by 12% SDS— PAGE. As shown, all fusion constructs (ECD, RI, Rl.l, R1.2,Rl.3, RI .4, RI .5, and R16) have robust protein expression. FIG. ZB depicts a western blot showing binding of an exemplary humanized anti-44 BB antibody to full length 4-lBB ECD fusion ptide, and R1 .1, RI 2, RI .3, and RI .6 fusion polypeptides, but not to the RI .4 or R1.S fusion polypeptides. lmmunoblots were performed with an exemplary anti—human 4-1BB antibody. Molecular size markers are indicated in kDa on the left. depicts binding afﬁnity of anti-4—IBB monoclonal antibodies for recombinant human 4-l BB antigen, as measured by ELISA. 0mm...“ values are represented on the y-axis, and increasing concentrations of anti—4-lBB antibodies (in ug/ml) along the x-axis.

BBK-4 (circles) is a murine anti—human 4-lBB antibody, 94G] (squares), 94K (upward pointing triangles), 94KV (diamonds), 94KVT (stars) and EUlOl (downward pointing triangles) are exemplary humanized variant antilBB antibodies. depicts binding of anti-4~]BB monoclonal antibodies to 4-1BB sing Jurkat T cells (Jurkat 8-1). Mean Fluorescence Intensity (MFI) values are represented on the y- axis and LoglO concentration of antibody (in ug/ml) along the . BBK—4 es) is a murine anti-human 4-IBB dy, 94G] (squares), 94K (upward pointing triangles), 94KV nds), 94KVT ) and EUlOl (downward pointing triangles) are exemplary humanized variant antiIBB antibodies. provides a table listing in l’ill'O binding aﬁiniti es of variant antil BB antibodies for 4-l BB. Binding afﬁnity was measured using surface plasmon resonance (SPR, Biacore 3000). 946] and EUIOI are exemplary humanized variant anti-4—lBB antibodies. ]0044] depicts binding of anti1BB monoclonal antibodies to 4-lBB expressing CD8- T cells. CD8+ T cells were isolated from human PBMCs and activated by anti-CD3 antibody for 2 days. 4-lBB-PE is an exemplary commercially available antiIBB antibody, BBK-4 is a murine anti-human 4-1BB antibody, 9401, 94K, 94KV, 94KVT and EUlOl are exemplary humanized variant anti.4-lBB dies. The graph in the bottom panel reﬂects the values shown for each antibody in the FACS data in the top panels. depicts a graph quantifying in vitro proliferation of CD8’ T cells treated with anti l BB antibodies. Proliferating CD8- T cells were treated with no antibody, human lgG alone, BBK-4, or an exemplary humanized t antilBB antibody: 9461, 94K, 94KV, 94KVT and EU101 and treated with WST-l -soluble olium salt) to stain proliferating (i.e., lically active) cells. depicts a graph quantifying in vilro IFNY secretion by CD8' T cells treated with - l BB antibodies. CD8“ T cells were isolated from human PBMCs and treated with no antibody, human IgG alone, or 1 ug/ml of an antiIBB antibody: BBK-4, 9401, 94K, 94KV, 94KVT and EUIOI. IFNy secretion was evaluated on days 1, 3, and 5. shows graphs depicting IFN-y secretion in (A) CD4- and (B) CD8‘. After being isolated from PBMCs of a healthy donor, activated T cells present in the PBMCs were rested in a RPMI-l 640+2%FBS medium for 24 hours, and the rested PBMCs were treated with an iron beads-attached anti-CD4 antibody or anti-CD8 antibody, and CD4‘ cells or CD8' cells were ed using an MACS magnetic separator. The isolated CD4' T cells or CD8’ T cells were d with a T cell activator, anti-CD3, to induce 4-lBB expression, and treated with EUlOl at ent concentrations (0.5, 1.0, 2.5, and 5,0 ug/ml) or a control human lgG (5.0 pg/ml) for 3 days. After 3 days, a culture medium excluding the cells was obtained, and fluorescence of human IFN-y in the culture medium was assessed by ELISA (ebioscience).

Results were compared with a standard curve as ed in an IFN-y ELISA kit.

A shows a graph depicted antibody-dependent cytotoxicity (ADCC) of exemplary anti-human 4-lBB antibodies BBK4, 94G], 94KVT and EU 10 l. 8 shows a graph depicting complement-dependent cytotoxicity (CDC) of ary anti-human 4-l BB antibodies BBK4, 9461, and EUlOl.

FIG. II shows in viva anticancer s of an exemplary uman 4-l BB antibody (EUl 0 l ) by concentration, which are measured as tumor sizes after colon cancer tumor cells (HT29) were subcutaneously injected into humanized mice, and when tumor sizes reach 100 to 200 mm", an exemplary anti-human 4-lBB antibody (EUlOl) was intravenously administered to mice at doses of 1.0 mg, 5.0 mg and 10.0 mg per 1 kg ofa body weight once every 5 days 3 times (representative data). shows anticancer effects of an exemplary anti-human 4- 183 antibody (EUlOl ) and an exemplary anti-PD-l antibody (Keytruda, “KD”) antibodies by concentration.

Anticancer effects were measured as tumor sizes after subcutaneous injection of colon cancer tumor cells (HT29) into humanized mice and antibody treatment. When tumor sizes reached 100 to 150 mm, mice were treated with an exemplary anti-human 4-l BB antibody (EUlOl ) or an exemplary anti—PD-l antibody uda) by intraperitoneal injection at a dose of 5.0 mg and .0 mg per 1 kg of body weight once every 5 days three times. ] shows comparative anticancer effects of individual ent and combination therapy of an exemplary uman 4-1BB antibody (EUIO l) and an exemplary anti-PD-l antibody (Keytruda). Anticancer effects were measured as tumor sizes aﬁer colon cancer tumor cells (HT29) were aneously injected into humanized mice and antibody treatment. When tumor sizes reach 300 to 450 mm}, an exemplary uman 4-] BB antibody (EUlOl) was administered at 2.5 mpk for individual treatment, an exemplary anti-PD-l antibody uda) was stered at 2.5 mpk for individual treatment, and EUIOI, 2.5 mpk + Keytruda, 2.5 mpk were stered for combination therapy. Administration was by intraperitoneal injection of mice, once every three days, for a total of three times.

A shows the numbers of human CD4* T cells and CD8? T cells circulated in mouse blood or I gram of tumor tissue at 34 days after treatment with an exemplary anti- human 4-lBB antibody (EUlOl) and an ary anti-PD-l antibody (Keytruda), individually and in combination, on tumor-implanted humanized mice, as described in . The number of T cell inﬁltrating lymphocytes (TILs) in tumor were measured by calculating tional ratios of the total cell numbers by measuring ratios (%) ofCD4’ T cells and CD8' T cells using a ﬂow cytometer. Flow cytometry was performed to measure the ratios (9/0) of the CD4' T cells and CD8’ T cells after cells are stained with a FITC-labeled CD4 antibody. a ﬂuorescent BVS 10- labeled CD8 antibody and a ﬂuorescent APC-cy7-labeled CD45 antibody, and a human blood cell marker, CD45-positive cells were separated from a ﬂow cytometry program (gating). 8 shows a ratio of Treg (CD4’Foxp3high T cells) per ratio of CDS‘IFN-y‘ T cells measured by calculating a proportional ratio between the ratio of the CDS‘IFN-y' T cells and the ratio of Treg (CD4Toxp3high T cells) using a ﬂow cytometer after the cells were stained with ﬂuorescent APC-cy7-labeled CD45, a ﬂuorescent BVS 10-labeled CD8 dy, a ﬂuorescent FlTC-labeled CD4 antibody, cent PE-labeled INFy, and ﬂuorescent APC- labeled Foxp3 antibody.

A and 3 show IFN-y analysis results through serum and tumor ﬂuid after individual and combination treatment of an exemplary anti-human 4—lBB antibody (EUlOl) and an exemplary anti-PD-l antibody (Keytruda). After dissection performed on all of the treated groups shown in FIGS. 15A and 15B, 10 pl of serum and [00 ul of tumor ﬂuid were analyzed with human IFN-y and human TGF-B ELISA kits.

A shows antigen-speciﬁc CD8’ T cell ratios (ratio of 4-1 BB’CD8‘ T cells: 43.2%, ratio of CD8’ T cells: 58.6%) measured before panning with an exemplary anti— human 4—lBB antibody (EUlOl ). 8 shows antigen-speciﬁc CD8’ T cell ratios (ratio of pCMV’CDS‘ T cells: 600%, ratio of CD8’ T cells: 79.3%) measured after panning with an exemplary anti-human 4-1BB antibody (EU | O 1 ).

CERTAIN DEFINITIONS In the description that follows, a number ofterms used in inant DNA and immunology are ively utilized. In order to provide a clearer and consistent understanding of the speciﬁcation and claims, including the scope to be given such terms, the following deﬁnitions are provided. |0056| About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will iate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a y of routes that may, in appropriate stances, be utilized for administration to a subject, for example a human. For example, in some embodiments, stration may be ocular, oral, parenteral, topical, etc.. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdemtal, transdemial, etc), enteral, intra-arten'al, intradennal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, hecal, intravenous, entricular, within a speciﬁc organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, aneous, sublingual, topical, al (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a ﬁxed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may e continuous dosing (e.g., ion) for at least a selected period of time.

Afﬁnity: As is known in the art, “afﬁnity” is a measure of the ess with a particular ligand binds to its partner. Affmities can be ed in different ways. In some embodiments, affinity is ed by a quantitative assay. In some such ments, binding r concentration may be ﬁxed to be in excess of ligand concentration so as to mimic physiological conditions. Alternatively or additionally, in some ments, binding partner concentration and/or ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under comparable conditions (e.g., concentrations).

Agom‘st: Those skilled in the art will appreciate that the term “agonist” may be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with an sed level or activity of another agent (i.e., the ed agent). In general, an agonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant activating activity. In some embodiments, an agonist may be direct (in which case it exerts its influence directly upon its ); in some embodiments, an agonist may be indirect (in which case it exerts its inﬂuence by other than binding to its ; e.g, by cting with a regulator of the target, so that level or activity of the target is altered).

Animal: as used herein refers to any member of the animal kingdom. In some embodiments, “cmimaf‘ refers to humans, of either sex and at any stage of development. In some embodiments, “animal” refers to non-human s, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or womis.

In some embodiments, an animal may be a transgenic animal, genetically engineered animal, and/or a clone. ] nist: Those skilled in the art will appreciate that the term “antagonist", as used herein, may be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with decreased level or activity of another agent (i.e., the inhibited agent, or target). In general, an antagonist may be or include an agent of any chemical class including, for e, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity. In some embodiments, an antagonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an antagonist may be indirect (in which case it exerts its inﬂuence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered).

Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements ient to confer speciﬁc binding to a particular target antigen. As is known in the art, intact antibodies as ed in nature are approximately ISO kD tetrameric agents comprised of two identical heavy chain ptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a ped” structure. Each heavy chain is comprised of at least four domains (each about I 10 amino acids long)— an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1, CH2, and the carboxy-tenninal CH3 ed at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions.

The “hinge” ts CH2 and CH3 domains to the rest of the antibody. Two disulﬁde bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody.

Each light chain is comprised of two s — an terminal variable (VL) domain, ed by a carboxy-tenninal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulﬁde bond; two other disulﬁde bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is . Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold“ formed from two beta sheets (e.g., 3-, 4-, or S- stranded ) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as ement determining regions" (CDRl, CDRZ, and CDR3) and four somewhat invariant “framework” regions (FRI, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three—dimensional space so that they create a single hypervariable antigen binding site d at the tip of the Y structure. The Fc region of naturally—occurring antibodies binds to elements of the complement system, and also to ors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, afﬁnity and/or other binding utes of Fe s for Fc receptors can be modulated through glycosylation or other modiﬁcation. In some embodiments, antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with d or engineered such glycosylation. For purposes of the present invention, in certain ments, any polypeptide or complex of polypeptides that includes sufﬁcient immunoglobulin domain sequences as found in natural dies can be referred to and/or used as an “antibody", whether such polypeptide is naturally produced (e.g, generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artiﬁcial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies, In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed ucts or s for utilizing antibody structural and functional features in altemative presentation, For e, ments, an antibody utilized in accordance with the present invention is in a format ed from, but not limited to, intact lgA, lgG, IgE or lgM antibodies; bi- or multi- speciﬁc antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab nts, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd nts, and ed CDRs or sets f; single chain Fvs; polypeptide-PC fusions; single domain antibodies (e.g., shark single domain antibodies such as lgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g, Probodies®); §mal| Modular immunoﬂharmaceuticals (“SM H’smi); single chain or Tandem diabodies (TandAb®); humabodies, VHHs; lins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR—like antibodies;, Adnectins®; Afﬁlins®; bodies®; Afﬁbodies®; TrimerX®; roteins; Fynomers®, Centyrins®; and KALBITOR®S. In some embodiments, an antibody may lack a covalent modiﬁcation (e.g., ment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modiﬁcation (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc] WO 27787 PCT/lBZlIlS/(Illll043 Antibodyfragment: As used , an ody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody nt may be produced by any means. For e, in some embodiments, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment may be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment may be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding dy fragment) may have a length of at least about 50, 60, 70, 80, 90, IOO, 110, 120, I30, I40, ISO, 160, I70, I80, 190 amino acids or more, in some embodiments at least about 200 amino acids.

Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct" binding involves physical contact between entities or es; indirect binding involves physical interaction by way of al contact with one or more intermediate entities. g between two or more entities can typically be assessed in any of a variety of contexts — including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

Cancer: The terms r”, “malignancy”, “neoplasm", “tumor”, and “carcinoma", are used herein to refer to cells that exhibit relatively al, uncontrolled, and/or autonomous growth, so that they t an aberrant growth phenotype characterized by a signiﬁcant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e. g., benign), malignant, pre-metastatic, metastatic, and/or tastatic . The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples ofdifferent types of s known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; as, melanomas, adenomas, carcinomas of solid tissue, squamous cell omas of the mouth, throat, latynx, and lung, liver cancer, genitourinary s such as prostate, al, r, e, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro—intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

CDR: as used herein, refers to a mentarity ining region within an antibody variable region. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR I CDRZ and CDR3, for each of the variable regions, A “set ofCDRs” or “CDR set” refers to a group of three or six CDRs that occur in either a single variable region capable of g the antigen or the CDRs of cognate heavy and light chain variable regions capable ofbinding the antigen. Certain systems have been established in the art for deﬁning CDR boundaries (e.g., Kabat, Chothia, etc); those skilled in the art appreciate the differences between and among these systems and are capable of understanding CDR boundaries to the extent required to understand and to practice the claimed invention.

Chemotherapeutic Agent: The term “chemotherapeutic agent”, has used herein has its art-understood meaning referring to one or more pro-apoptotic, atic and/or cytotoxic agents, for example cally including agents utilized and/or recommended for use in treating one or more es, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e. g. microtubule targeting agents such as taxanes, maytansine and analogs f, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.o., inhibitors of topoisomerase I and/or topoisomerase 11), one or more kinase inhihitors, one or more nucleotide analogs or tide precursor analogs, one or more peptide antibiotics, one or more platinum— based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more ofthe following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of mycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, omib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, hosphamide, Curcumin, Cytarabine, ubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, yurea, icin, Imatinib, Irinotecan, Maytansine and/or analogs thereof (e. g. DMl) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a sinoid, Oxaliplatin, Paclitaxel, exed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincn'stine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in the t of an antibody-drug conjugate. In some embodiments, a herapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: ML 1 -doxorubicin, hRS7-SN-3 8, hMN-l4-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7-ProP-Dox, hMN-l4-ProP-Dox, hLLZ-Pro-Z-P-Dox, hA20-Pro—2-P-Dox, hPAM4-Pro—2-P-Dox, hLLl- Pro-Z-P-Dox, P4/DlO-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR34I9, SARS66658, BIIBO l 5, BT062, SGN-75, SGN-CD19A, AMG-l72, AMG-595, BAY9343, ASG-SME, ASG-22ME, M8F, MDX-1203, MLN—0264, anti-PSMA ADC, RG—7450, RG-7458, RG—7593, RG- 7596, RG—7598, RG—7599, RG-7600, RG-7636, ABT-4l4, [MON-853, [MON-529, vorsetuzumab tin, and lorvotuzumab mertansine.

Combination therapy: As used herein, the term “combination y” refers to those situations in which a subject is aneously exposed to two or more therapeutic ns (e.g., two or more therapeutic agents). In some embodiments, the two or more eutic regimens may be administered simultaneously. In some embodiments, the two or more therapeutic regimens may be administered sequentially (e.g., a first regimen stered prior to administration of any doses of a second regimen). In some embodiments, the two or more therapeutic regimens are administered in overlapping dosing ns. In some embodiments, administration of combination therapy may involve administration of one or more therapeutic agents or modalities to a subject receiving the other s) or modality.

Carr-espomling to: As used herein, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For e, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identiﬁed as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for es of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding lo" a residue at position 190, for example, need not actually be the 190m amino acid in a particular amino acid chain but rather corresponds to the e found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to fy “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment gies, ing re programs such as, for example, BLAST, CS— BLAST, CUSASWH, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMJVIER, HHpred/l-II-Isearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPI—II, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in ptides and/or nucleic acids in accordance with the present disclosure.

Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered” when the polypeptide sequence manipulated by the hand of man. For example, in some embodiments of the present invention, an engineered polypeptide comprises a sequence that includes one or more amino acid ons, deletions and/or insertions that have been introduced by the hand of man into a reference polypeptide sequence. Comparably, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously t has been introduced, for example by transformation, mating, c ization, transfection, transduction, or other mechanism, or previously present genetic material is d or removed, for example by substitution or deletion mutation, or by mating ols). As is common practice and is understood by those in the art, tives and/or progeny of an engineered polypeptide or cell are typically still referred to as “engineered“ even though the actual manipulation was performed on a prior entity.

Epitope: as used herein, includes any moiety that is specifically recognized by an immunoglobulin (cg, antibody or receptor) binding component. In some embodiments, an epitope is comprised of a ity of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

Ex vivo: as used herein refers to biologic events that occur outside of the context of a multicellular organism. For example, in the context of cell-based systems, the term may be used to refer to events that occur among a population of cells (e.g., cell proliferation, cytokine ion, etc.) in an ial environment. [0073| Framework ework region: as used herein, refers to the ces of a variable region minus the CDRs. Because a CDR sequence can be determined by different systems, likewise a framework sequence is subject to pondingly different interpretations.

The six CDRs divide the framework regions on the heavy and light chains into four sub-regions (FRl, FR2, FR3 and FR4) on each chain, in which CDR] is positioned between FRI and FR2, CDR2 between FRZ and FR3, and CDR3 between FR3 and FR4 Without specifying the particular sub-regions as FRI, FR2, FR3 or FR4, a framework region, as ed by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, FRl, for example, represents the first framework region closest to the amino terminal end of the variable region and 5' with respect to CDR], and FRs represents two or more of the sub-regions constituting a framework region.

Humanizerl: as is known in the art, the term “hmmmized’ is commonly used to refer to antibodies (or antibody components) whose amino acid sequence includes VH and VL region sequences from a reference dy raised in a non-human species (e.g., a mouse), but also es modifications in those sequences relative to the reference antibody intended to render them more “human-like", i.e., more similar to human getmline variable sequences. In some embodiments, a “humanized” antibody (or antibody ent) is one that immunospecifically binds to an antigen of interest and that has a framework (FR) region having substantially the amino acid sequence as that of a human dy, and a complementary determining region (CDR) having substantially the amino acid ce as that of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a man immunoglobulin (i.e., donor immunoglobulin) and all or ntially all of the framework s are those of a human immunoglobulin consensus sequence. In some embodiments, a zed antibody also comprises at least a portion of an globulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a zed antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include a CH1, hinge, CH2, CH3, and, optionally, a CH4 region of a heavy chain constant region.

In vitro: The term “in vilro” as used herein refers to events that occur in an artiﬁcial environment, e.g., in a test tube or reaction , in cell culture, etc, rather than within a multi-cellular organism. |0076] In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a man animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vilro systems).

Isolated: as used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced er in nature and/or in an experimental setting), and/or (2) designed, produced, ed, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered led” or even , after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be “isolated’ when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in ation with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is sized in a cellular system different from that which produces it in nature is considered to be an “isolated‘ polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more puriﬁcation ques may be ered to be an “isolated” polypeptide to the extent that it has been separated from other components a) with which it is associated in ; and/or b) with which it was associated when initially produced. '1).- as used herein, refers to the dissociation constant of a binding agent (e.g., an antibody or binding component thereof) from a complex with its partner (e.g., the epitope to which the antibody or binding component thereof binds).

Operably linked: as used herein, refers to ajuxtaposition wherein the components described are in a relationship permitting them to function in their ed manner. A control t "operab/y linked" to a functional element is associated in such a way that expression and/or ty of the functional element is achieved under ions compatible with the control element. In some ments, "operably linked“ control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in from to or ise at a from the functional element of interest.

Pharmaceutical composition: As used , the temt “pharmaceutical composition“ refers to a ition in which an active agent is formulated together with one or more phamtaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically signiﬁcant probability of achieving a predetermined therapeutic effect when administered to a nt population.

Polypeptide: The term “polypeptide", as used herein, generally has its artrecognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that n sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, oﬂen greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually assing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modiﬁcations or analogs known in the art. Useful modiﬁcations include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, ically active portions f, and/or characteristic portions thereof.

Prevent or prevention: as used herein when used in tion with the occurrence of a disease, er, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset and/or ty of one or more characteristics or symptoms of the disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a tion susceptible to the disease, disorder, or ion.

Recombinant: as used , is intended to refer to polypeptides that are ed, engineered, prepared, sed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides sed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, atorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been lated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed. created or isolated by any other means that involves splicing or ligating selected nucleic acid ce elements to one another, chemically synthesizing ed ce elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silica. In some embodiments, one or more such selected ce elements results from mutagenesis (e.g., in vivo or in viiro) ofa known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).

Speciﬁc binding: As used herein, the term “speciﬁc binding" refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to “bind .speciﬁc‘a/bl” to the target with which it interacts. In some embodiments, speciﬁc binding is assessed by ing or detennining degree of association between the binding agent and its partner; in some embodiments, speciﬁc binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, speciﬁc binding is assessed by detecting or ining ability of the binding agent to compete an ative interaction between its r and another entity. In some embodiments, speciﬁc binding is assessed by performing such detections or determinations across a range of concentrations.

Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is ing from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, er, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or ion. In some ments, a subject is someone with one or more features teristic of susceptibility to or risk of a disease, disorder, or condition. In some ments, a subject is a t. In some embodiments, a subject is an individual to whom diagnosis and/or y is and/or has been administered.

Therapeutic agent: As used herein, the phrase peutic agent” in general refers to any agent that elicits a d cological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically signiﬁcant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be deﬁned by s criteria, such as a certain age group, gender, genetic background, preexisting clinical ions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features ofa e, disorder, and/or condition. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be ed by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical iption is required for stration to humans.

Therapeutically Effective : As used herein, the term “therapeutically effective amount” means an amount that is sufficient. when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically ive amount” does not in fact require successful treatment be achieved in a particular dual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to ts in need of such treatment. For example, in some embodiments, term “therapeutically effective amount”, refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, ate, or reverse a cancer-supportive s occurring in said dual, or will enhance or increase a cancensuppressive process in said individual. In the context of cancer treatment, a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. A particularly preferred “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy such as a pancreatic carcinoma or helps achieve or prolong remission of a malignancy. A eutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a therapeutically effective amount administered to promote remission or inhibit asis. As with most cancer therapies, the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure“ for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to effect a desirable or beneficial change in the health of an dual who has cancer. Such beneﬁts are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further asis, a decrease in opportunistic ions, an increased survivability, a decrease in pain, improved motor function, improved cognitive ﬁmction, improved feeling of energy (vitality, decreased e), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and ations thereof In addition, regression of a particular tumor in an individual (e.g., as the result of treatments described herein) may also be assessed by taking s of cancer cells from the site of a tumor such as a pancreatic adenocarcinoma (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to r the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype. For example, tumor regression d by employing the methods of this invention would be indicated by finding a decrease in any of the pro-angiogenic markers sed above, an increase in anti-angiogenic markers bed herein, the normalization (i.e., alteration toward a state found in normal individuals not suffering from cancer) of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer. Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or stered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or stered in a plurality of doses, for example, as part of a dosing regimen.

Variant: As used herein in the context of molecules, e.g., nucleic acids, ns, or small molecules, the term “variant“ refers to a molecule that shows signiﬁcant structural identity with a reference molecule but differs structurally from the nce molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural ty with the reference molecule. As will be appreciated by those skilled in the art, any ical or chemical reference molecule has certain characteristic structural elements. A variant, by deﬁnition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a ptide may have a characteristic sequence element comprised ofa plurality of amino acids having designated ons relative to one another in linear or three-dimensional space and/or buting to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or tide sequence. In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94915, 95‘3”, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic ce element with a reference ptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or c acid.

Vector: as used herein, refers to a nucleic acid molecule capable of transporting another c acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.

Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., ial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. er, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA, ucleotide synthesis, and tissue culture and transformation (e.g., electroporation, ction). Enzymatic reactions and puriﬁcation techniques may be performed according to manufacturer's speciﬁcations or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more speciﬁc references that are cited and discussed throughout the present speciﬁcation. See e.g., Sambrook et al.. Molecular Cloning: A tory Manual (2“ ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989)), which is incorporated herein by reference for any purpose.

DETAILED DESCRIPTION OF ARY EMBODIMENTS [0090'] The present disclosure relates, inter alia, to 4- l BB, which is an ble co- stimulatory molecule, and eutic antibodies that bind thereto that have been engineered to have improved characteristics over a reference antilBB antibody. For e, engineered antibodies provided herein have been d to enhance antigen affinity relative to that of a reference agonist antibody that speciﬁcally recognizes an epitope within the extracellular domain of human 4-1BB (Korean Patent No. 10-0500286, Accession No: KCTC ). Speciﬁcally, as described herein, the inventors engineered a reference humanized anti-human 4-IBB antibody, 9461 (US Patent No. 045). As described es herein, the light chain and heavy chain CDR sequences of a reference antibody 94Gl, were separately engineered to improve the afﬁnity of each chain. Moreover, as described herein, exemplary engineered anti lBB antibodies can effectively induce eration of activated T cells. Notably, ary engineered antil BB antibodies are capable of inducing surprisingly ed activity of CD8- T cells due to the stimulation caused by the 4-lBB humanized antibody binding to a 4- 188 molecule and inhibiting activation-induced cell death (AICD). Thus the present disclosure es engineered anti-human 4—1BB antibodies with improved properties over a reference antibody, and moreover demonstrate that these dies have surprisingly beneﬁcial activity in vitro and in viva. 4-1BB (also referred to as CD137, TNFRSF9, etc) is a receptor belonging to the tumor necrosis factor or (TNFR) superfamily. 4-lBB is a co-stimulatory molecule generally expressed in activated T lymphocytes and involved in immunity and autoimmune diseases (Kwon et al. PNAS84:2896,1987; Kwon et al. PNAS (1989) 86: I963; Son et al. Journal Q/‘Immzmological Methods (2004) 286(1-2):187-201, each of which is herein incorporated by reference in its entirety). Human 4-1 BB is a 255 amino acid protein (Accession No.

NM_001561; NP_001552). The complete human 4~1BB amino acid sequence is provided in SEQ ID NO: 44. 4-lBB is expressed on the cell surface in monomer ('30 kDa) and dimer (55 kDa) forms and likely trimerizes with 4-1BB ligand to signal.

Current tanding of 4- 18B suggests that it is constitutively expressed on a number of cells, albeit at low levels, including Foxp3’ Tregs and dendritic cells (DC). (See, Vinay and Kwon (2014) BMB Rep. 47(3): 9, which is incorporated by reference herein.) tion with a number of agonists, such as cytokines (e.g., 1L-2, IL—4), polyclonal activators (cg, Con A and PHA), cell surface molecules (e. g., anti-CD3 and anti-CD28) and promoters of Caz- ion and PKC activity (e.g., ionomycin and photbol myristate acetate) further enhance expression of 4-1BB. Id.

Numerous studies of murine and human T cells indicate that 4-1BB promotes enhanced cellular proliferation, survival, and cytokine production (Croft, 2009, Nm. Rev. lmmzmol. 9:271-285). Studies have indicated that some 4-1 BB agonist monoclonal antibodies can increase costimulatory molecule expression and markedly enhance cytolytic T lymphocyte responses, resulting in anti-tumor efﬁcacy in s models. 4-lBB agonist monoclonal antibodies have demonstrated efficacy in prophylactic and therapeutic settings. Further, 4—lBB monotherapy and combination therapy tumor models have established durable anti-tumor protective T cell memory ses (Lynch (2008) l. Rev. 22: 277—286). 4-1BB agonists also have been shown to t autoimmune reactions in a y of art-recognized autoimmunity models (Vinay .]. Mol. Med. 84:726-736). This dual activity of 4-1BB offers the potential to provide anti-tumor ty while dampening autoimmune side effects that can be associated with immunotherapy approaches. 4-] BB antibodies and nts thereof The present sure provides, at least in part, engineered anti-human 4-] BB antibodies and fragments f that exhibit markedly, and unexpectedly, superior characteristics in vitro and/or in viva. For example, certain provided antibodies have increased y relative to a reference humanized uman 4-‘l BB antibody. |009Sl In some embodiments, an anti I BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 heavy chain CDR sequences that are or include a sequence of SEQ ID NOS: 5 to 8. In some embodiments, an anti1BB antibody or antigen-binding dy fragment includes one or more of: a heavy chain CDR] that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8. In some embodiments, an anti lBB antibody or antigen-binding antibody fragment includes each of: a heavy chain CDRI that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8.

In some embodiments, an antil BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 light chain CDR sequences that are or include a sequence of SEQ ID NQs: 1-4. In some embodiments, an -1BB dy or antigen-binding antibody fragment includes one or more of: a light chain CDR] that is or includes a sequence of SEQ ID NO: I, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4. In some embodiments, an -IBB antibody or antigen-binding antibody fragment includes each of: a light chain CDR] that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4. |0097| In some ments, an anti l BB antibody or antigen—binding antibody nt includes a heavy chain variable domain that includes a heavy chain CDRI that is or includes a sequence of SEQ ﬂ) NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8 and/or a light chain variable domain that includes a light chain CDR] that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 4.

In some embodiments, an antiIBB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 where the 5'h amino acid, asparagine (N), was substituted with glutamine (Q), glutamic acid (E) or serine (S). In some embodiments, an anti—4-1BB dy or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 where the 5”] amino acid, asparagine (N), was substituted with valine (V), glycine (G), or proline (P).

In some embodiments, an anti IBB antibody or antigen-binding dy nt includes a light chain le domain that es a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4 where the 6‘h amino acid position of LCDR3 is mutated.

In some embodiments, an -IBB antibody or antigen-binding antibody fragment es a heavy chain le domain that includes a heavy chain framework I (FRI) region comprising a sequence of SEQ ID NO: l6 or 17. In some embodiments, an antiIBB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain ork 3 (FR3) region comprising a sequence of any one of SEQ ID NOs: 18-20. In some embodiments, an anti1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework 1 (FRI) region comprising a sequence of SEQ ID NO: 16 or 17 and a heavy chain framework 3 (FR3) region comprising a ce of any one of SEQ ID NOs: 18-20.

In some embodiments, an anti lBB antibody or n-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOS: I 1-14.

In some embodiments, an anti-4—IBB dy or antigen-binding antibody fragment includes a heavy chain variable domain that is or es a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID N05: 1 1-14. In some embodiments, an antiIBB antibody or antigen- binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOS: 1 l-I4.

In some embodiments, an antilBB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a light chain variable domain that has or includes a ce of SEQ ID NO: 9 or 10. In some embodiments, an anti—4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.39" of SEQ ID , 99.4% or 99.5% identical to a ce NO: 9 or 10. In some embodiments, an anti1BB antibody or n-binding antibody fragment includes a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10.

In some embodiments, an anti1BB antibody or n-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: I 1-14 and a light chain variable domain that is or includes a ce of SEQ ID NO: 10. In some embodiments, an anti1BB dy or antigen-binding antibody nt includes a heavy chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID NOS: 1 1-14 and a light chain le domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence ofSEQ ID NO: 10. In some embodiments, an antilBB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence ed from SEQ 11) NOS: 1 1-14 and a light chain variable domain that is or includes a sequence of SEQ ID NO: 10.

Amino acid sequences of anti-human 4-1BB antibody or antigen-binding fragment binds of the present disclosure may be substituted through conservative tution.

The term “conservative substitution" used herein refers to modiﬁcation of a ptide in which one or more amino acids are substituted with an amino acid having a similar biochemical property so as not to cause the loss of a biological or biochemical function of the corresponding polypeptide. The term “conservative ce variant” or “conservative amino acid substitution” used herein is the substitution of an amino acid residue with an amino acid residue having a r side chain Amino acid residues having a similar side chain are deﬁned in the art. Those residues encompass amino acids with a basic side chain (e.g., lysine, arginine, and histidine), amino acids with an acidic side chain (e.g., aspartic acid and glutamate), amino acids with a non-charged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with a non-polar side chain (cg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with a beta- branched side chain (e. g., threonine, , and isoleucine) and amino acids with an ic side chain (e.g., tyrosine, phenylalanine, tryptophan, and ine). Therefore, it is expected that the antibody of the t invention can have conservative amino acid substitution, and still ensure an activity.

In some embodiments, an uman 4-1BB antibody or antigen-binding fragment of the present disclosure may include a constant region selected from an IgGl constant domain, an IgG2 constant domain, an IgGl/IgG2 hybrid constant , a human IgG4 constant domain, an IgA nt domain, an IgE constant domain, an lgM constant domain, and an IgD constant domain.

In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is or es an IgA, IgD, IgE, lgM, IgG, or variants thereof.

In some embodiments, an anti-human 4- I BB antibody of the present disclosure includes a t Fc-region that has an amino acid mutations and/or tutions at one or more positions of 234, 235, 236, 237, 238, 239, 253, 254, 265, 266, 267, 268, 269, 270, 288, 297, 298, 299, 307, 311, 322, 327, 328, 329, 330, 331, 332, 434 and 435.

In some embodiments, an anti-human 4-lBB dy or antigen-binding fragment of the present disclosure is human IgG] isotype. In some embodiments, an anti-human 4-IBB antibody or antigen-binding fragment of the present disclosure includes a variant IgGl.

In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes an IgGl polypeptide that has amino acid mutation at one or more positions of 233, 234, 23 5, 236, 265, 297, 329, 33] and 322.

In some embodiments, an anti-human 4-1 BB antibody or antigen—binding fragment includes an IgGl polypeptide containing one or more mutations in L234, L235, D270, N297, E318, K320, K322, P331 and P329. In some embodiments, an anti—human 4-lBB antibody or antigen-binding fragment es an IgGl polypeptide containing two, three, four, or more mutations in L234, L235, D270, N297, E318, K320, K322, P331 and P329. In some WO 27787 PCT/lBZOlS/(Nﬂl043 embodiments, an anti—human 4-lBB dy or antigen-binding fragment includes an IgGl polypeptide with mutations in L234A and L235A. |0110| In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes a light chain constant region. In some embodiments, an uman 4-lBB antibody or antigen-binding fragment includes a kappa (K) and/or lambda 0») light chain and/or a variant f.

In some embodiments, an anti-human 4-l BB antibody or antigen-binding fragment is a monoclonal antibody. In some embodiments, an anti-human 4-IBB antibody or antigen-binding fragment is a Fab fragment, a Fab' nt, a F(ab')2 fragment, a Fv fragment, a disulftde—bonded Fv fragment, a scFv fragment, a single domain antibody, humabody, nanobody, and/or a diabody. In some embodiments, an anti—human 4—lBB antibody or n- binding fragment is a monovalent antibody. In some ments, an anti-human 4-lBB antibody or antigen-binding nt is a multivalent antibody. In some embodiments, an anti- human 4-lBB antibody or antigen-binding fragment is a multi-speciﬁc antibody (e.g., a bispeciﬁc antibody).

In some embodiments, the present disclosure encompasses methods of modifying the carbohydrate content of an antibody of the disclosure by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and encompassed within the disclosure, see, e.g., US. Pat. No. 6,218,149; EP 0 359 096 B 1; US.

Publication No. US 2002/0028486; W0 835; U.S. Publication No. 2003/01 l5614; US.

Pat. No. 6,218,149; US. Pat. No. 6,472,51 1; all of which are incorporated herein by nce in their entirety. In other embodiments, the present disclosure asses methods of modifying the carbohydrate content of an antibody of the present disclosure by deleting one or more endogenous carbohydrate moieties of the antibody. In a specific embodiment, the present disclosure encompasses deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine. In some ments, an uman 4- 188 antibody or antigen-binding fragment ses a N297A mutation in the CH2 domain. In some embodiments, the N297A mutation results in aglycosylation, which reduces FcR or C lq binding. In some embodiments, an anti-human 4-lBB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region sing a N297A mutation and a K322A mutation. In some etnbodiments. an anti-human 4—IBB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising a N297A mutation and a 0265A mutation. In some embodiments, an anti-human 4-IBB antibody or antigen-binding nt comprises a heavy chain comprising an Fc region comprising a N297A mutation, at 0265A mutation, and a K322A mutation. In some ments, an anti—human 4—lBB antibody or antigen-binding fragment ses an Fc region with a L234A mutation and/or a L23 5A mutation. In some embodiments, an anti-human 4-1 BB antibody or antigen-binding fragment comprises an Fc region with one or more mutations selected from L234A—, L23 5A, N297A, D265A, and K322A. In some ments, an anti—human 4-1BB antibody or antigen-binding fragment comprises Fc region with two or more mutations selected from L234A—, L23 5A, N297A, D265A, and K322A. In some ments, an anti-human 4-lBB antibody or antigen- binding fragment comprises Fc region with three, four, or five mutations selected from L234A~, L235A, N297A, D265A, and K322A. |0113| Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycofonns may be generated by any method known to one skilled in the art, for example by using engineered or variant expression s, by co-expression with one or more enzymes, for example Dl N- acetylglucosaminyltransferase III (GnTIl 1), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying ydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 172176-180; Davies et al., 20017 Biotechnol Bioeng 742288-294; Shields et a1, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) US. Pat. No. 6,602,684; US. Ser. No. 10/277,370; US. Ser. No. 10/] 13,929; PCT W0 00/61739A1; PCT W0 01/292246A1; PCT W0 02/31 1 140A]; PCT W0 54A1; POTILLEGENTTM technology (Biowa, Inc. Princeton, N.J.); ABTMglycosylation engineering technology RT biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., W0 39; EA01229125; US 200301 15614; Okazaki et a1., 2004, JMB, 336: 1239-49 each of which is incorporated herein by reference in its entirety.

In some ments, an anti-human 4— 1 BB antibody or antigen—binding fragment of the present disclosure is as an agonist for human 44 BB.

In some embodiments, an anti-human 4-1BB antibody or antigen—binding fragment of the present sure binds to a human 4-1 BB molecule. In some embodiments, an anti-human 4-IBB antibody or antigen-binding fragment of the present disclosure specifically binds to a human 4-1 BB molecule.

In some embodiments, an anti-human 4- 1 BB antibody or antigen-binding fragment binds to a sequence that is or includes that of SEQ ID NO: 15. In some embodiments, an anti-human 4-lBB dy or antigen-binding fragment binds to an epitope of 4-lBB extracellular domain that is or es a sequence of SEQ ID NO: 15. |01|7| In some embodiments, binding of an anti-human 4-IBB antibody or antigen- binding fragment of the present disclosure with human 4-1 BB extracellular domain is abrogated by one or more mutations of SEQ ID NO: 44 selected from N30, D38, N39, R41, A56, G57, R60 or T61, In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-l BB molecule with a binding affinity (KB) of lx lO‘7 to lxlO'” M. In some embodiments, an anti-human 4-lBB antibody or n- binding fragment of the present disclosure binds to a human 4-l BB molecule with a binding afﬁnity (KD) of lxlO'“ to 1x 10'12 M. Binding affinity (KD) may be measured, for e, by surface plasmon resonance, for example, using a BIACORE .

In some embodiments, an anti-human 4-1BB antibody or antigen-binding nt of the present disclosure binds to a human 4-lBB le or a fragment thereof at a binding affinity (KB) of less than l,0><10'R M. In some embodiments, an anti-humanized 4- lBB antibody or antigen-binding fragment of the present disclosure binds to a human 4-lBB molecule or a nt thereof at a binding affinity (KD) of less than 1.0><IO"’ M. In some embodiments, an anti-humanized 4- lBB antibody or n-binding fragment of the present disclosure binds to a human 4-lBB molecule or a fragment f at a binding affinity (K0) ofless than LONG |0120| In some embodiments, an anti—4- 1 BB antibody or antigen-binding fragment of the present disclosure fails to bind or weakly binds a non-primate 4—lBB polypeptide (e.g., a canine, mouse and rat 4~IBB polypeptide). In some embodiments, an anti1BB antibody or antigen— binding fragment of the present sure binds efﬁciently to human or monkey 4-lBB. This binding affinity suggests that the structure and/or sequence of epitope for a primate 4-IBB antibody may be quite different from canine, mouse and rat. |0121| In some embodiments, an anti-human 4-lBB antibody or n-binding fragment of the present disclosure is an agonistic antibody. In some embodiments, an anti- human 4-lBB antibody or antigen-binding fragment of the present disclosure mediates T cell activation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds CD8? and/or CD4“ T cells expressing human 4-IBB_ In some embodiments, an anti-human 4-lBB antibody or antigen-binding fragment of the present disclosure does not have or has low ADCC activity. In some embodiments, an anti-human 4- IBB antibody or antigen-binding fragment of the present disclosure does not have or has low CDC activity. In some embodiments, an anti-human 4—l BB antibody or antigen-binding fragment of the present disclosure does not have or has low ADCC ty and CDC activity. In some embodiments, an anti-human 4-1BB antibody or antigen- binding fragment of the present disclosure has an ADCC cell killing activity of less than about less than about 20%, less than about l0°/o, less than about 8%, or less than about 5%. In some embodiments, an anti-human 4-l BB antibody or antigen-binding fragment of the present disclosure has an ADCC cell killing activity of less than about l0°/o. In some embodiments, an anti-human 44 BB antibody or antigen-binding nt ofthe t sure has a CDC cell killing ty of less than about 30%, less than about 20%, less than about 109/6, less than about 8%, or less than about 59/6. In some embodiments, an uman 4-IBB dy or antigen-binding fragment of the present disclosure has a CDC cell killing activity of less than about 20%.

In some embodiments, an uman 4-lBB antibody or antigen-binding fragment of the present disclosure is characterized by low toxicity (e.g, a low degree of post administration cell death). In some ments, an anti-human 4-lBB antibody or antigen- binding fragment of the present disclosure is characterized by low hepatoxicity. In some ments, a subject that has been administered an anti—human 4-IBB antibody or antigen- binding fragment of the present disclosure at a therapeutic dose has levels of one or more of ALT, AST and total bilirubin in a normal range. In some ments, an uman 4-1BB antibody or antigen-binding fragment ofthe present disclosure is characterized by an ability to treat ts for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, can contribute to the therapeutic results ed. "Low immunogenicity" is deﬁned herein as raising signiﬁcant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (Elliott et al., Lancet 344:] 125—1 127 (1994), entirely incorporated herein by reference).

Nucleic Acids The sure provides polynucleotides comprising a nucleotide sequence encoding uman 4-lBB antibodies of the present disclosure and fragments thereof. Anti- human 4-IBB antibodies and nts thereof as described herein may be produced from nucleic acid molecules using molecular biological methods known to the art. Nucleic acids of the present disclosure include, for example, DNA and/or RNA.

In some embodiments, nucleic acid constructs include regions that encode an anti- human 4-1BB antibody or fragment thereof (e.g., 94K, 94KV, 94KVT, EUlOl). In some embodiments, such antibodies or fragments thereof will include VH and/or VL regions. An anti- human 4-1BB antibody or fragment f may be identiﬁed and/or selected for a desired binding and/or functional properties, and variable regions of said dy isolated, amplified, cloned and/or ced. Modiﬁcations may be made to the VH and VL nucleotide sequences, including additions of nucleotide sequences encoding amino acids and/or canying restriction sites, and/or substitutions of nucleotide sequences encoding amino acids. In some embodiments, a nucleic acid sequence may or may not include an intron sequence.

Where appropriate, nucleic acid sequences that encode anti-human 4- 1 BB dies and fragments thereof (e.g., 94K, 94KV, 94KVT, EUlOl) may be modified to include codons that are optimized for expression in a particular cell type or organism (e.g., see US.

Patent No. 5,670,356 and US. Patent No. 5,874,304). Codon optimized sequences are tic sequences, and preferably encode the identical polypeptide (or a biologically active nt of a full length polypeptide which has substantially the same activity as the full length polypeptide) d by the non-codon optimized parent polynucleotide. In some embodiments, the coding region of the genetic material encoding dy ents, in whole or in part, may include an altered sequence to optimize codon usage for a particular cell type (e.g., a eukaryotic or prokaryotic cell). For example, a coding ce for a humanized heavy (or light) chain variable region as described herein may be optimized for expression in a bacterial cells.

Alternatively, the coding sequence may be optimized for expression in a ian cell (_e.g., a CHO cell). Such a sequence may be described as a codon-optimized sequence.

Nucleic acid constructs of the present disclosure may be inserted into an expression vector or viral vector by methods known to the art, and nucleic acid molecules may be ly linked to an expression control sequence. A vector comprising any of the above- described nucleic acid molecules, or fragments thereof, is further provided by the present disclosure. Any of the above nucleic acid molecules, or nts thereof, can be cloned into any suitable vector and can be used to transform or transfect any suitable host. The selection of vectors and methods to construct them are commonly known to persons of ry skill in the art and are described in general technical references (see, in general, binant DNA Part D,” Methods in Enzymology, Vol. 153, Wu and Grossman, eds, Academic Press (1987)).

In some embodiments, conventionally used techniques, such as, fore example. electrophoresis, calcium phosphate precipitation, extran transfection, lipofection, etc. may be used to introduce a foreign nucleic acid (DNA or RNA) into a prokaryotic or otic host cell. Desirably, a vector may include regulatory ces, such as transcription and translation initiation and ation codons, which are speciﬁc to the type of host (e.g., bacterium, ﬁmgus, plant or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA. In some embodiments, a vector comprises regulatory sequences that are c to the genus of the host. Preferably, a vector comprises tory sequences that are speciﬁc to the species of the host.

In addition to the replication system and the inserted nucleic acid, a c acid construct can include one or more marker genes, which allow for selection of ormed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an ophic host to provide prototrophy, and the like.

Suitable vectors include those designed for propagation and expansion or for expression or both. For e, a cloning vector is selected from the group consisting of the pUC series, the pBluescript series (Stratagene, LaJolla, Calif), the pET series (Novagen, Madison, Wis), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif). Bacteriophage vectors, such as XGTIO, kGTl l, kZapII (Stratagene), XEMBL4, and KNM] 149, also can be used. Examples of plant expression vectors include pBll 10, pBllOl .2, pBIlOl .3, pBIlZl and pBlNl9 ech). Examples of animal expression vectors include pEUK-C l and pMAMneo (Clontech). The TOPO cloning , pMAM system (lnvitrogen, Carlsbad, Calif.) also can be used in ance with the manufacturer's recommendations.

An sion vector can comprise a native or nonnative promoter operably linked to an isolated or puriﬁed nucleic acid molecule as described above. Selection of promoters, e.g., strong, weak, inducible, -speciﬁc and developmental-specific, is within the skill in the art. Similarly, combining of a nucleic acid molecule, or fragment thereof, as bed above with a promoter is also within the skill in the art.

Suitable viral vectors include, for example, retroviral vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, AAV—adenoviral chimeric vectors, and adenovirus—based vectors, and lentiviral vectors, such as Herpes x (HSV)-based vectors. These viral vectors can be prepared using standard inant DNA techniques described in, for example, Sambrook et al., lar Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY. (1994).

A retroviral vector is derived from a retrovirus. Retrovims is an RNA virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into the genome of its host cell and is ated along with host cell DNA, thereby constantly ing viral RNA and any nucleic acid sequence incorporated into the retroviral genome. As such, long-term sion of a therapeutic factor(s) is achievable when using irus.

Retroviruses contemplated for use in gene therapy are relatively non-pathogenic, although pathogenic retroviruses exist. When employing pathogenic retroviruses, e.g., human immunodeﬁciency virus (HIV) or human T-cell lymphotrophic s (HTLV), care must be taken in altering the viral genome to eliminate toxicity to the host. A retroviral vector additionally can be manipulated to render the virus replication-deft cient. As such, iral vectors are considered particularly useful for stable gene transfer in viva. Lentiviral vectors, such as HIV-based vectors, are exemplary of retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non- dividing cells and, therefore, can be of use in ng persistent forms of disease.

Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of g vectors, expression vectors, rs, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra). [0135'] In some ments, nucleic acids and vectors of the present disclosure may be ed and/or purified. The present disclosure also provides a composition comprising an described isolated or d nucleic acid molecule, optionally in the form of a vector.

Isolated nucleic acids and vectors may be prepared using standard techniques known in the art including, for example, alkali/SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis and other ques well known in the an. The composition can comprise other components as described further herein.

In some embodiments, nucleic acid molecules are inserted into a vector that is able to express an anti-human 4-lBB antibody or fragment thereof when introduced into an appropriate host cell. Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Exemplary host cells e prokaryotes (e.g., I}. coli) and eukaryotes (e.g., a COS or a CHO cell). Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C 127 cells, Cos l, Cos 7 and CV l, quail QCl-3 cells, mouse L cells and e hamster ovaiy (CHO) cells (e.g., DG44 cells). In some embodiments, a mammalian host cell suitable for the expression of the antibody may be a Chinese Hamster Ovary ('CHO) cell (for example, ing DHFR-CHO cells used along with a DHFR-selectable marker), an NSO myeloma cell, a COS cell or an SP2 cell.

Any (s) known to one skilled in the art for the insertion of DNA fragments into a vector may be used to uct expression vectors encoding an anti-human 4-lBB antibody or fragment thereof of the present disclosure under control of transcriptional/ translational control s. These methods may include in vitro recombinant DNA and synthetic techniques and in viva recombination (See, e.g., Ausubel, supra; or Sambrook, supra).

Production of Antibodies Antibodies and n-binding nts of the t invention may be prepared and/or puriﬁed by any technique known in the art, which allows for the subsequent formation ofa stable antibody or antibody fragment.

A nucleic acid ng an anti-human 4-l BB antibody and/or antigen-binding fragment of the present disclosure may be easily ed and sequenced by conventional procedures. For example, an ucleotide primer designed to speciﬁcally amplify ponding heavy chain and light chain-coding regions from a hybridoma or phage template DNA may be used. ed nucleic acids may be inserted into an expression vector, and then desired monoclonal antibodies may be produced from a suitable host cell (that is, transformant) transformed by introducing the expression vector to the host cell. In some embodiments, a method for preparing anti -human 4-1 BB antibody and/or antigen—binding fragment of the present sure may include amplifying an expression vector including a nucleic acid encoding the antibody, but is not d thereto.

In some embodiments, a host cell is eukaryotic host cell, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, antibodies and antibody fragments of the t disclosure can be glycosylated or can be non-glycosylated. In some embodiments, a recombinant expression vector encoding an anti-human 4-lBB antibody and/or antigen-binding fragment of the t disclosure is introduced into a mammalian host cell and an antibody may be prepared by culturing the host cell for a sufﬁcient time to s the antibody. In some embodiments, a mammalian host cell is cultured for a sufficient time to secrete an antibody or antibody fragment of the present disclosure in a culture medium.

In some embodiments, an sed antibody of the present disclosure may be uniformly puriﬁed after being isolated from the host cell. Isolation and/or ation of an antibody of the present disclosure may be performed by a conventional method for isolating and purifying a protein. For example, not wishing to be bound by theory, an anti-human 4—lBB antibody and/or antigen—binding fragment of the present disclosure can be recovered and puriﬁed from inant cell cultures by well-known methods including, but not limited to, protein A puriﬁcation, protein G puriﬁcation, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, afﬁnity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ") can also be employed for puriﬁcation. See, e.g., Colligan, Current Pro/owls in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each entirely incorporated herein by reference. In some embodiments, an antibody of the present disclosure may be isolated and/or puriﬁed by additionally combining ﬁltration, superﬁltration, salting out, dialysis, etc.

Puriﬁed anti-human 4-IBB antibodies and/or antigen-binding fragments of the present disclosure can be characterized by, for example, ELISA, T, ﬂow cytometry, immunocytology, BlACORETM analysis, SAPIDYNE KINEXATM c exclusion assay, SDS- PAGE and Western blot, or by HPLC analysis as well as by a number of other functional assays disclosed herein.

Therapeutic Applications The present disclosure encompasses a recognition that engineered anti-human 4- lBB antibodies and antigen-binding fragments may be useful for sis, prevention, and/or treatment of certain diseases such as, for example, cancer. Any of the antilBB antibodies or antigen-binding fragments provided herein may be used in therapeutic methods. For example, an antil BB antibody or antigen-binding fragment of the present disclosure can be used as immunotherapeutic agents, for example in the treatment of a malignant disease (e.g, cancer).

The present disclosure provides methods for ng and/or preventing a malignant disease, said methods including administering an anti1BB antibody or antigen- binding fragment of the t disclosure to a subject. s for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, include, but are not limited to, cancer and/or and the treatment of matory diseases.

Cancer treatments in the context of the present disclosure may be mediated through increasing xic T cells and anti-cancer cytokines. Generally, antigen-speciﬁc cell- mediated immunity is caused by cytotoxic T cells, and includes two signaling : a ﬁrst signaling event is induced when a T cell recognizes an antigen from an antigen-presenting cell via a or, and a second signaling is d by co—stimulatory les. Due to the ﬁrst and second stimuli, T cell activity and related factors are increased, thereby g T cells speciﬁcally functioning in cancer ent, and the formed T cells are increased in cytotoxicity, cell division, cell viability and anti-cancer cytokine secretion due to stimulation with the co- stimulatory molecules.

Speciﬁcally, it has been demonstrated that stimulation by 4-lBB can e the activity of CD8' T cells, increase secretion of anti-cancer cytokines such as interferon gamma (IFNy), se expression of anti-apoptotic molecules such as Bel-2, BchL and Bfl—l, and/or inhibits activation-induced cell death (AICD). In some embodiments, an anti-human 4-lBB antibody or antigen-binding fragment of the present disclosure can enhance or increase one or more of CD8‘ T cell activity, secretion of anti-cancer nes such as interferon gamma (lFNy), expression of anti-apoptotic molecules such as Bel-2, BchL and Bﬂ-l, and inhibition of activation-induced cell death (AICD). In some embodiments, therapeutic treatment with an anti- human 4-lBB antibody or antigen-binding nt of the present sure can reduce and/or inhibit growth of cancer cells.

In some embodiments, the present disclosure provides a method for delaying or inhibiting tumor growth, comprising regulation of cytokine secretion in vivo or in vilro by administering an anti-human 4-1 BB antibody or antigen-binding fragment of the present disclosure. l n some embodiments, the present disclosure es a method for reducing tumor burden, comprising regulation of cytokine secretion in vivo or in vilro by administering an anti- human 4- 1 BB antibody or antigen-binding fragment of the present disclosure.

In some embodiments, the t disclosure provides a method for treating cancer or tumor by monitoring to a biological subject of cancer or tumor to be treated, sing: (i) strating an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure to a subject, (ii) separating then isolating a biological sample from the t, (iii) measuring a secretion amount of INFy or TGFB from the sample and ting a proportion ratio and (iv) ining a therapeutically effective amount of the antibody or antigen-binding fragment thereof by comparing the control samples which are administrated or not administrated with the anti-human 4-lBB antibody or antigen-binding fragment thereof.

In some embodiments, the present disclosure provides a method of treating a subject in need f, the method comprising a step of administering to the subject a composition that comprises or delivers an antilBB dy or antigen-binding fragment of the present disclosure and/or a nucleic acid the same. In some embodiments, a subject has or is at risk for developing cancer. In some ments, the present disclosure provides a method W0 27787 PCT/lBZOIS/(llltl043 for preventing or treating cancer or tumor of a patient, which includes administering a therapeutically effective amount of the humanized 4-lBB antibody or the antigen-binding fragment thereof to a patient with cancer or tumor.

In some ments, the present disclosure provides a method of inducing an immune response in a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or delivers an antilBB antibody or antigen-binding fragment of the present disclosure and/or a nucleic acid the same. In some embodiments, a subject has or is at risk for ping cancer. [0151[ In some embodiments, the present disclosure provides a method of enhancing an immune response or increasing the activity of an immune cell in a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or rs an anti 1 BB antibody or n-binding fragment of the present disclosure and/or a nucleic acid the same. In some embodiments, a subject has or is at risk for developing cancer.

Cancers le for treatment with method of the present disclosure can e, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal , head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, elioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, a cancer for ent with an antiIBB antibody or antigen-binding fragment of the present disclosure may include, but is not d to, oma, lymphoma (e.g., Hodgkin’s and non-Hodgkin’s lymphomas), blastoma, sarcoma and leukemia. In some embodiments, cancer may include squamous cell carcinoma, small cell lung cancer, all cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer. pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal , endometrial or uterine carcinoma, salivary oma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other proliferative disorders, and s types of head and neck cancer.

A composition including an antilBB antibody or antigen-binding fragment of the present disclosure may be administered at a ceutically effective amount to treat cancer cells or metastasis thereof, or inhibit the growth of cancer. For use in eutic methods, an anti1BB antibody or antigen-binding fragment of the present disclosure would be formulated, dosed, and stered in a fashion consistent with good l ce. Factors for consideration in this context e the particular disorder being treated, the particular mammal being d, the clinical condition of the individual patient, the age of the patient, the weight of the patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. |0154| The present disclosure es high aﬁ'mity anti-human 4-lBB dies that may have superior properties relative to a reference antibody. The present disclosure encompasses a recognition that these antibodies may have improved ability to induce T cell activation and/or secretion of cytokines such as IFNy. Accordingly, the present disclosure encompasses a recognition that an anti-human 4-lBB antibody or antigen binding fragment of the present sure may be administered a dose lower than reference antibody.

In some embodiments composition that includes an antilBB antibody or antigen—binding fragment of the t disclosure may be administered to a patient as a bolus or by continuous injection when . In some embodiments, bolus administration is of an anti- 4-1 BB Fab of the present sure and may be administered at a dose of 0.0025 to 100 mg/kg, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg, or 0.10 to 0.50 mg/kg. 1n the case ofthe continuous injection, the antibody of the present invention presented as a Fab nt may be administered at a dose of0.001 to 100 mg/kg/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min or 0.10 to 0.50 mg/kg/min for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours. In some embodiment, an antibody of the present disclosure is a full~length antibody (having a complete constant domain). In some embodiments, a full-length antibody is administered at a dose of approximately 0.01 to 10 mg/kg, 1 to 8 mg/kg, or 2 to 6 mg/kg. In some embodiments, a full-length antibody is administered by injection for 30 to 35 s. Administration ncy may vary depending on the severity of a condition. For example, the frequency may be once every 2 to 7 days, once a week, or once every I, 2, 3 or 4 weeks.

In some embodiments, a composition may be administered to a patient by subcutaneous injection. Specifically, the antibody may be administered to a patient at a dose of 0.1 to 100 mg by aneous injection once every 2 to 7 days, every week, once every two weeks, or every month.

Combination Therapies The present disclosure provides therapeutic methods that include administration of an anti-human 4-1BB dy or antigen-binding fragment of the present disclosure in ation with one or more other therapies.

In some embodiments, an anti-human 4—lBB antibody or antigen-binding fragment is administered in combination with one or more therapies that have been approved for treatment of cancer. For example, combination treatment of with an anti 1 BB antibody and a conventional chemotherapeutic, cisplatin, has been shown to have synergistic activity in tumor killing and prevention of organ-speciﬁc toxicity—. (Kim et al., Cancer Research (2008) 68( l 8): 7264-9) In some embodiments, an anti-human 4-IBB antibody or antigen-binding fragment of the present disclosure is administered in combination with a second therapy selected from an immune checkpoint inhibitor, eukin 12 (IL-12), Granulocyte-macrophage colony- ating factor (GM-C SF), an anti-CD4 agent, and a chemotherapeutic agent, such that the subject receives treatment with both.

In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the t disclosure is administered to a t that been administered or will be administered a composition comprising a chemotherapeutic agent, such that the t receives treatment with both. Therapeutic methods of the present disclosure may include stration of any chemotherapeutic agent known in the an. In some ments, chemotherapeutic agent is stered to a subject that been administered or will be administered a composition comprising an anti-human 4-lBB antibody or antigen—binding fragment.

In some embodiments, an anti-human 4-1BB antibody or antigen—binding fragment is stered to a subject that been administered or will be administered a composition comprising ﬂuorouracil. In some embodiments, fluorouracil is administered to a subject that been administered or will be administered a composition comprising an anti—human 4-1BB antibody or antigen-binding fragment. In some ments, an anti-human 4-lBB antibody or antigen-binding fragment is stered to a subject that been administered or will be administered a composition comprising doxorubicin. In some embodiments, doxorubicin is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-lBB dy or antigen-binding fragment. In some embodiments an anti-human 4-IBB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising irinotecan. In some embodiments, irinotecan is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-IBB antibody or antigen—binding fragment. In some ments, an anti-human 4-IBB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising paclitaxel. In some ments, paclitaxel is administered to a subject that been administered or will be stered a composition comprising an anti-human 4—IBB antibody or antigen-binding nt, In some embodiments, an uman 4-lBB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising cisplatin. In some embodiments, cisplatin is administered to a subject that been administered or will be administered a composition sing an anti-human 4-lBB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-IBB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition sing cyclophosphamide. In some embodiments, cyclophosphamide is administered to a t that been administered or will be stered a composition comprising an anti-human 4— 1 BB dy or antigen-binding fragment.

In some embodiments, an anti-human 4-IBB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition sing GM-CSF, such that the subject receives treatment with both. In some embodiments, GM-CSF is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-lBB antibody or antigen-binding fragment.

In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be W0 27787 PCT/lBZOlS/(NIO043 administered a composition comprising IL-IZ, such that the subject receives treatment with both.

In some embodiments, IL-12 is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-IBB antibody or antigen-binding fragment.

In some embodiments, an anti—human 4-lBB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been stered or will be administered a composition comprising an anti-CD4 agent, such that the subject receives treatment with both, In some ments, an anti-CD4 agent is administered to a t that been administered or will be administered a composition comprising an anti-human 4~lBB dy or antigen-binding fragment.

In some embodiments, an anti-human 4-]BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition comprising a checkpoint inhibitor (e.g., an immune checkpoint inhibitor), such that the subject receives treatment with both. In some embodiments, an immune oint inhibitor is administered to a t that been administered or will be administered a composition comprising an uman 4-] BB antibody or antigen-binding fragment.

A checkpoint tor used in ation with an anti-human 4—1BB antibody or anti gen-binding fragment of the present disclosure can be, for example, any immune checkpoint inhibitor. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7- H4, BTLA, CTLA—4, CD277, IDO, KIR, PD-l, LAG-3, TIM-3, TIGIT and VISTA. An immune checkpoint inhibitor may refer to any compound that ts the function of an immune inhibitory checkpoint protein. Inhibition includes ion of function and full blockade. In some embodiments, an immune checkpoint inhibitor is an antibody that speciﬁcally recognizes an immune checkpoint protein. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint n inhibitors, ative immune checkpoint inhibitors may be developed in the (near) future. Immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molecules and small molecules.

In some embodiments, an immune checkpoint inhibitor is an inhibitor of CTLA— 4. In some embodiments, a checkpoint inhibitor is an antibody that targets CTLA—4, such as, for example, ipilimumab. In some embodiments, a checkpoint inhibitor targets CD366, which is a transmembrane protein also known as T cell immunoglobulin and mucin domain containing protein—3 (TIM-3). In some embodiments, an immune checkpoint inhibitor is an agent that inhibits PD-l signaling. [0l69l PD-l (i.e. programmed cell death n-l), is a protein that is distributed on the surface of an immune cell such as a T or B cell and is also known as CD279. In a human, PD—l is expressed by a PDCDI gene located at the 2p37.3 position on chromosome 2. PD-l is known to bind two ligands, PD-Ll and PD-LZ.

In some embodiments, an D-l agent is administered to t who is receiving, has received or will e treatment with an anti-human 4-] BB antibody or antigenbinding fragment of the t disclosure. In some certain embodiments, an anti-human 4-IBB antibody or antigen-binding fragment of the present disclosure is administered to patient who is receiving, has received or will receive treatment with an anti-PD-l agent.

In some embodiments, an anti-PD-Ll agent is administered to patient who is receiving, has received or will receive treatment with an anti-human 4—IBB antibody or antigenbinding fragment of the present disclosure. In some certain embodiments, an anti-human 4-] BB antibody or antigen-binding fragment of the present disclosure is administered to patient who is receiving, has received or will receive treatment with an anti-PD-Ll agent. In some embodiments, agents that inhibit PD-Ll include, for example, AMP-244, 736, MPDL328 0A, MIl-Il.

In some embodiments, an anti-PD-l agent is an agent that inhibits PD-l. In some embodiments, an anti-PD-l agent is an agent that inhibits PD-Ll and/or PD-LZ. In some embodiments, an antibody agent that ts PD-I signaling is a onal antibody or a fragment f. In some embodiments, an antibody agent that inhibits PD~1 signaling is an anti~PD~l antibody or fragment thereof.

In some ments, an anti-PD-I antibody is administered to patient who is receiving, has ed or will receive treatment with an anti-human 4-l BB dy or n— binding fragment of the present disclosure. In some certain embodiments, an uman 4—IBB antibody or antigen—binding fragment of the t disclosure is administered to patient who is receiving, has received or will receive treatment with an anti-PD-l antibody. Anti-PD~I antibodies include, for example, nivolumab, pembrolizumab, atezolizumab, durvalumab, and W0 27787 PCT/lBZOIS/(l00043 avelumab. olizumab (Keytruda, Merck) is an antibody therapeutic that inhibits PD-I activity.

As described in the Examples of the present application, administration of an anti- human 4-lBB antibody or antigen-binding fragment of the present disclosure in ation with an anti-PD-l antibody may enhance efﬁcacy relative to either treatment alone, and further may also reduce tionally known side effects.

In some certain embodiments, pembrolizumab is administered to patient who is receiving, has received or will receive ent with an anti-human 4—l BB antibody or antigenbinding fragment ofthe present disclosure. In some certain embodiments, an anti—human 4-l BB antibody or antigen-bi nding fragment of the present disclosure is administered to patient who is receiving, has received or will e ent with pembrolizumab.

In some embodiments, an immune checkpoint tor (cg, an anti-PD-l agent) is administered to a patient in an amount of from about 0.01 mg/kg to about 100 mg/kg. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD-I agent) is administered to a patient in an amount within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 005 mg/kg, 0.075 mg/kg, 0.] mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, I mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. In some embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, l mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, l0 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or l00 mg/kg. In some embodiments, an immune checkpoint inhibitor (e.g., an anti—PD-l agent) may be administered to a patient in an amount of from about I mg/kg to about 20 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 4 mg/kg, from about 3 mg/kg to about 5 mg/kg, or from about 3 mg/kg to about 4 mg/kg. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD—l agent) may be administered to a patient in an amount of about I mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, or about 5 mg/kg.

In some embodiments, treatment with a ation of an immune checkpoint inhibitor and an anti-human 4-IBB antibody or n-binding fragment of the present disclosure may e proliferation, ion, persistence and/or cytoxic activity of CD8‘ T cells in a subject.

Cell-based Applications Yet another object of the present invention is to provide a method for proliferating activated T cells ex vivo by administering the 4- I BB humanized antibody or antigen-binding fragment thereof.

In some embodiments, a method for ex viva proliferation and/or isolation of activated T cells includes contacting a population of T cells with an antilBB antibody or anti gen-binding fragment of the present disclosure, thereby increasing proliferation of activated T cells.

In some embodiments, a method for erating activated T cells ex vivo includes stering an antiIBB antibody or antigen-binding fragment of the present disclosure. In some embodiments, activated T cells are proliferated and/or ed from a sample of eral blood mononuclear cells (PBMC). PBMCs can be obtained/isolated using methods known in the art.

In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes administration of an D3 monoclonal antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). [n some ments, a method for ex vivo proliferation and/or isolation of activated T cells includes administration of IL~2 and/or IL-IS to the culture medium (e.g., at concentration that is at least about l0 units/ml). |0182'] In some embodiments, a method for isolating antigen—speciﬁc activated T cells includes (a) culturing peripheral blood mononuclear cells (PBMC) in a medium together with a peptide of an epitope of interest and lL—2; (b) inducing 4-lBB expression in the ed cells by adding the e of the epitope ofinterest; (c) contacting the cultured cells with a surface coated with an anti1BB antibody or antigen—binding fragment, wherein cultured cells expressing 4- 188 adhere to the coated surface; and (d) removing unattached cells, thereby isolating antigen-speciﬁc activated T cells.

In some embodiments, the activated T cells are CD8- T cells.

In some embodiments. lymphocytes (e.g., T cells) are cultured at a temperature of at least about 25 °C, preferably at least about 30 °C, more preferably about 37 °C.

The present disclosure asses the recognition that activated T cells (e. g., CD8" T cells). generated by the methods described herein may be therapeutically useful (veg, for the treatment of ).

Cell-based ies [0186‘] The present disclosure provides methods to selectively isolate and mass culture CD8' T cells which recognize an autologous cancer antigen (self-tumor antigen), for example, an autologous cancer n that overexpressed in cancer cells while present in a low ratio in normal cells. The present sure that cells (e.g., CD8- T) isolated by these methods may be useful for the treatment of cancer.

In some embodiments, a method for treating and/or preventing cancer in a subject in need thereof includes administering to the subject a therapeutically effective amount of activated T cells produced by an ex vim method such as those described herein.

Upon appropriate reactivation, tumor antigen speciﬁc T cells can recognize and eliminate gous tumor cells. For example, tumor antigen speciﬁc T cells can be generated ex vivo using methods as bed herein. Upon adoptive transfer, specifically reactivated T cells from cancer patients can efﬁciently reject autologous human tumors in viva.

The t disclosure provides methods for preventing and/or ng cancer and/or tumor of a patient, which include administering a therapeutically effective amount of activated T cells prepared ex vim by administering an anti1BB antibody or antigen-binding fragment of the present sure.

In some embodiments, T cells for using in a therapeutic method are nic (from the same species but different donor) as the recipient subj ect. In some embodiments, T cells for using in a therapeutic method are autologous (the donor and the recipient are the same).

In some embodiments, T cells for using in a therapeutic method are syngeneic (the donor and the recipients are different but are identical twins).

VI’1! In some embodiments, the cells are formulated by ﬁrst harvesting them from their e medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin.

A treatment-effective amount of cells in the composition is at least l0“, typically greater than 108, at least 109 cells, and generally more than low. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For e, if cells that are speciﬁc for a ular antigen are desired, then the population will contain greater than 70%, lly greater than 80%, 85% and 90-95% of such cells. For uses ed herein, the cells are lly in a volume of a liter or less. In some embodiments, cells for administration are in a volume of less than 500 ml, less than 250 ml, or 100 ml or less. In some embodiments, a density of the desired cells is typically greater than 10" ml and generally is greater than [07 cells/ml, generally 108 ml or greater. A clinically relevant number ofimmune cells can be apportioned into multiple infusions that cumulatively equal or exceed 108 cells, l0° cells, 10'0 cells. 10ll cells, or 10[2 cells.

Compositions Provided herein are compositions comprising dies and antigen binding fragments that speciﬁcally bind to an epitope of a human 4- 13B polypeptide. Compositions of the present disclosure (eg, compositions that r an anti -human 4-] BB antibody or antibody fragment) may include any suitable and effective amount of a composition for use in delivering 3 provided anti-human 4- 138 antibody or antibody fragment to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. Also provided herein are compositions that include activated cell populations (e.g., activated T cell population) that have been ted via a method of the present disclosure (e.g., a method that includes a step contacting a cell with an anti-human 4-lBB antibody or antibody fragment).

W0 27787 PCT/[BZOIS/(NNNH3 Compositions of the present disclosure include ceutical compositions that include an anti—human 4-lBB antibody or antigen-binding nt disclosed herein and/or a cell population obtained by a method disclosed herein. In some embodiments, a pharmaceutical composition can include a buffer, a diluent, an excipient, or any combination thereof. In some ments, a composition, if desired, can also contain one or more additional therapeutically active substances.

In some ments, an antiIBB dy, n-binding fragment and/or cell population of the present disclosure are suitable for administration to a mammal (e.g., a human). Although the descriptions of pharmaceutical compositions provided herein are principally ed to ceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to s of all sorts. Modiﬁcation of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologi st can design and/or perform such modiﬁcation with merely ordinary, if any, experimentation.

In some embodiments, compositions are formulated for parenteral administration.

For example, a pharmaceutical composition provided herein may be provided in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection or intravenous infusion), For example, in some embodiments, a pharmaceutical compositions is provided in a liquid dosage form that is suitable for injection. In some embodiments, a pharmaceutical composition is provided as powders (e.g., lyophilized and/or sterilized), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, buffer, salt solution, etc.) prior to injection.

In some embodiments, a pharmaceutical composition is diluted and/or tituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, ate buffered saline, etc. In some embodiments, a powder should be mixed gently with the aqueous diluent (e.g., not ). ]0197'] In some embodiments, an antilBB antibody, antigen-binding fragment, and/or cell tion of the present disclosure is formulated with a phannaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer‘s solution, dextrose solution, and l— 10% human serum albumin. Liposomes and nonaqueous es such as fixed oils can also be used. A vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, ol) and al stability (e.g., buffers and preservatives) In some embodiments, a formulation is sterilized by known or suitable techniques.

Formulations of the phamiaceutical compositions described herein may be prepared by any method known or ter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a diluent or another excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or ing the product into a desired single- or multi- dose unit.

In some embodiments, a pharmaceutical composition including an antiI BB antibody, antigen-binding fragment, and/or cell population of the present disclosure can be included in a container for storage or administration, for e, an vial, a syringe (e.g., an IV e), or a bag (e.g., an IV bag). A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit close” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. ve amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ients in a ceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject d and further depending upon the route by which the composition is to be administered. The examples below describe, in part, dosing of an exemplary anti-human 4—lBB antibody to a rodent. Standard methods are known in the art of how to scale dosing in animal systems. See, for example, J Basic (‘lin Pharm. March 2016-May 2016; 7(2): 27—3 1, which is incorporated herein by reference in its entirety. By way of example, the composition may se n 0.1% and 100% (w/w) active ingredient.

In some embodiments, a composition comprises or delivers an anti-human 4-1BB dy or antigen-binding fragment of the present disclosure at a dose of 0.0] mg/kg to 100 mg/kg. In some embodiments, a composition comprises or rs an anti-human 4-IBB WO 27787 PCT/lBlelS/(lllll043 antibody or antigen-binding fragment at a dose in an amount within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.] mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. In some embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.] mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, IO mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg. |0202| A pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, es any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, e active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The e and Practice of Pharmacy, let n, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known ques for the preparation thereof. Except insofar as any tional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a rious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. |0203| In some embodiments, a ceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or [00% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating . surface active agents and/or emulsiﬁers, disintegrating agents, binding agents, vatives, buffering agents, lubricating , and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and itory waxes, coloring agents, g , sweetening, ﬂavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.

In some embodiments, a provided pharmaceutical composition ses one or more phannaceutically acceptable excipients (e.g., vative, inert diluent, dispersing agent, surface active agent and/or emulsifier, buffering agent, etc). In some embodiments, a pharmaceutical composition comprises one or more preservatives. In some embodiments, pharmaceutical compositions comprise no preservative. |0206| In some embodiments, a composition including an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is stably formulated. In some embodiments, a stable formulation of an anti-human 4-1BB antibody or antigen-binding nt of the t sure may comprise a ate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi—use preserved formulations le for pharmaceutical or veterinary use. Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p- cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and osal, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include, no preservative, 0.1—29/0 m—cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 01-396 benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5., 1.9, 2.0, 2.5%), 0.001-0.5°/o thimerosal (e.g., 0.005, 0.01), 0001-2094: phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1 .0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 00075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

In some embodiments, a pharmaceutical composition is provided in a form that can be refrigerated and/or . In some embodiments, a pharmaceutical composition is ed in a form that cannot be refrigerated and/or frozen. In some embodiments, reconstituted solutions and/or liquid dosage forms may be stored for a certain period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, a month, two months, or ). In some embodiments, storage of antibody itions for longer than the specified time results in antibody degradation.

Liquid dosage forms and/or reconstituted solutions may comprise particulate matter and/or discoloration prior to administration. In some embodiments, a solution should not be used if discolored or cloudy and/or if particulate matter remains after ﬁltration.

General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 2151 ed, Lippincott Williams & s, 2005.

The present disclosure further provides a pharmaceutical pack or kit comprising one or more containers ﬁlled with at least one anti-human 4-lBB antibody or antibody fragment as described . Kits may be used in any able method, including, for example, therapeutic methods, diagnostic methods, cell proliferation and/or isolation methods, etc.

Optionally ated with such container(s) can be a notice in the form ibed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or ical ts, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both.

In some embodiments, a kit may include one or more reagents for detection (e.g, detection of an anti-human 4-1BB antibody or antibody fragment). In some embodiments, a kit may include an anti-human 4-lBB antibody or dy fragment in a detectable form (e. g., covalently associated with detectable moiety or entity).

In some embodiments. an anti~human 4-1 BB antibody or antibody fragment as provided herein may be included in a kit used for treatment of subjects. In some embodiments, an anti-human 4-lBB antibody or dy fragment as provided herein may be included in a kit used for proliferation and/or isolation of T cells (e.g., CD8° T cells).

The contents of all cited references (including literature references, issued patents, published patent ations, and co-pending patent applications) cited throughout this ation are hereby expressly incorporated by reference.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments. However, the following examples are merely provided to illustrate the t invention, but the scope of the present invention is not limited to the following examples.

EXAMPLES The present disclosure es, at least in part, humanized anti-human 4-] BB antibodies and nts thereof with ed properties that contain one or more ural features that are not found in a reference humanized anti human 4-IBB antibody, 94Gl. 94Gl was generated by humanization of the murine anti human 4-lBB antibody BBK-4 antibody.

Antigen-recognizing sites (CDR regions) were determined using CDR loop assignment (lMGY': Lefranc, 1997) and a 3—D model (Swiss—de Viewer (www.cxpasyorg». A phage display library was prepared with diversity in a total of 10 sites ing 4 sites on the amino acid sequence of the light chain and 6 sites of the heavy chain was constructed. After panning, approximately 14 humanized antibody clones out of 1,000 clones were selected (for a total of six humanized scFvs), and among the selected clones, 94Gl, was obtained (Son et al. J. Immunol. thods (2004) 286: 187-201). These humanized antibodies, including 94Gl, had afﬁnities for human 4-IBB n that were less than H IO'h that of BBK-4, but were active in l’i/I'O, The present disclosure encompassed the recognition that structural variants of 9461, may have improved properties. Generation and characterization of t humanized anti-human 4-]BB antibodies and fragments thereof is described in further detail in the following examples Example I —— Preparation of humanized anti-human 4-] BB antibodies This example describes the production of an ary anti-human 4-] BB antibodies with improved affinity over a reference 94G] antibody. 94G] was generated by humanizing a murine anti human 4-lBB antibody (BBK 4) as described in Son et al. J. Immunol.

Mel/10d; (2004) 286: l87-20 l, which is herein incorporated by reference in its entirety. Also used herein is a H4-lBB antigen (Accession No: KCTC ) that is speciﬁcally ed from activated T cells (e.g., activated T cell line), and has not been identiﬁed from unstimulated T cells. For e, a H4-1BB antigen can be isolated from T cells that have been matured by phorbol myristate acetate (PMA), ionomycin, Concanavalin A, or anti CD3i, This H4-lBB antigen has a size of 1.4 kb, and 60% gy with mouse 4-lBB (GarnioWagner et al., Cellular logy (I996) 169: 91-98, which is herein incorporated by reference in its entirety). In this example, 9401 was divided into a light chain and a heavy chain vectors, each of optimized to generate ed humanized antibodies. l0217| The present disclosure encompasses a recognition that a suitable method for generating improved humanized anti—human 4-lBB antibodies or fragments thereof is through single, stepwise amino acid substitutions and/or combinations thereof. The present disclosure es various structural variants of humanized anti-human 4-l BB antibodies and fragments thereof with one or more structural features (e.g., amino acid substitutions) that are not found in a 940] dy. The t disclosure further encompasses a recognition that structural features can be combined for stepwise improvements in one or more antibody properties (e.g, increased antigen afﬁnity).

First, a humanized uman 4-lBB antibody with increased afﬁnity relative to 94G] nce antibody was obtained by ng a CDR region of a light chain, rather than a heavy chain. This light chain structural variant was ﬁxed, and combined with zed anti- human 4-lBB antibody heavy chains structural variants with, e.g., mutations in the CDR region of 946l . Further structural features were integrated to generate humanized anti-human 4-l BB antibodies with high afﬁnity and/or other improved characteristics. 1.] Cons/merit)” of ors |0219] Vectors with a 940] light chain and 9461 heavy chain, respectively, were constructed by changing pComb3H-HA to be sed in a Fab type to improve a heavy chain and a light chain of a humanized antibody in E. coli (.l. Immuno/ Mel/rods (2008) 329(1-2):176- 83; Virology (2004) 318: 598). Speciﬁcally, a 940] light chain was inserted into a vector designed by changing an APZ tag (SEQ ID NO: 42 — NANNPDWDFNP) with a flag tag (SEQ ID NO: 43 - DK), the ﬂag tag is designed to be located downstream thereof, and has a human heavy chain sequence (Accession No. ABOIQ438) obtained from known data of the NCBI GenBank was placed as a constant domain in a heavy chain position. In addition, after a 94G] light chain sequence was cloned into the vector, it was transferred to Eco/i (e.g., TGl) (F' [traD36 proAB+lachlacZAM15]supE thi-l A(lac-proAB) A(mch- hstM)5, (rK-mK-) by transformation, followed by selection of a transformed vector called pCOM-Fab—94G l -L, which was used as a backbone to induce afﬁnity tion of the light chain (Table I). The above- described method was similarly d out for the 94G] heavy chain, and a ed vector was called pCOM-Fab-94Gl-H. An improved light chain, 94/w, was designed as the light chain of pCOM-Fab-94G l, which served as backbone for production of heavy chain variants with improved afﬁnity.

Table l - 94G! and 94/w LCDR amino acid sequences SEQ ID NO: I QTISDY LCDR l SEQ ID NO: 3 QDGHSFPPT LCDR 3 SEQ ID NO: 4 QDGHSWPPT LCDR 3.6 variant 94/w I. 2 Aﬂinily maturation ofhumanized uman 4-]BB antibody lighl chain |0221| Described herein is the development a humanized anti-human 4-lBB antibody with a light chain variant that has improved binding afﬁnity. An antibody with a high affinity was obtained by changing LCDR3 (SEQ ID NO: 3) ofa 94G] light chain in the context of the ab-94Gl-L vector described above as follows. Various DNA sequences encoding a light chain were amplified by PCR using primers [using NNS (N: A, T, C, G; S: C, 0)] designed to insert 19 different amino acids into each amino acid position of the 9 amino acids SEQ ID NO: 3, constituting the LCDR3 part of the 946] light chain. Ampliﬁed products were ligated to a light chain position of the vector and then transformed into Eco/i TO]. All clones with light chain structure variants of LCDR3 were substituted in different forms and collected to prepare nine position mixes. To assess whether each amino acid position was substituted with a ent amino acid, two clones changed in respective positions were randomly chosen and analyzed by sequencing using an ABl-3 730xl sequencer. which showed that the amino acid residues at tive positions were substituted at various positions.

To see r 94G] Fab variants with mutations at different LCDR3 positions had increased antibody ty, each position mix was expressed by adding IPTG (to a ﬁnal concentration of lmM) to Eco/i T01, and then Fab antibody present in a supernatant was ted to ELISA. Speciﬁcally, each on mix was cultured with shaking in a 2YT medium in a 37 °C incubator until the culture had an absorbance at 600 nm of 0.8 or more, then overnight cultured at 30 °C with IPTG (e.g., at a ﬁnal concentration of 1 mM). ELISA was performed the following day on a supernatant obtained by centrifugation at 12,000 rpm for 10 minutes at 4 °C. Binding afﬁnities were determined for the various 94G] LCDR3 variant Fabs by dividing the binding activities of each clones with respect to 4-lBB Fab by the expression levels for the respective mutant clone. A 94G| LCDR3 variant with a mutation position 6 of LCDR3 .6) showed the t binding affinity.

Subsequently, to determine how various mutations of 94Gl at the LCDR3.6 position impacted antibody afﬁnity, 25 monoclonal antibodies were isolated from pCOM- Fab94GI-LCDR3.6 position mix and expressed by adding IPTG (e.g., at a ﬁnal concentration of 1 mM) to E. coli (e.g., TG] ), cultured, and ELISA was performed on a Fab antibody t in a supernatant. Binding afﬁnities were determined for the various 94G] LCDR3.6 clones by dividing the 4-lBB Fab binding activities of each clones by the sion levels for each.

A 94G] LCDR3.6 variant with phenylalanine at the LCDR3.6 position substituted with tryptophan exhibited the highest binding afﬁnity. A Fab dy prepared by substituting the constant heavy chain of pCOM-Fab94Gl-L with the heavy chain of the backbone 9461 on the ed 940] light chain was called 94/w. Thus, a 94/w t includes an improved 94G] light chain in which the 6“1 amino acid of LCDR3 is substituted with tryptophan (W) (QDGHSWPPT — SEQ ID NO: 4) and a 94G] heavy chain. IPTG-induced expression in Eco/i and ELISA ofa 94/w Fab was used to determine binding affinity as described above. Using this method, it was ined that a 94/w Fab antibody has a binding ty 3.5 times higher than that of 9461 (Fab antibody) (data not shown).

I. 3 Aﬂiiiity maturation ofhumanized anti-human 4- IBB antibody hearty chain CDRs‘ Described herein is the pment humanized anti-human 4- l BB antibodies with heavy chain structural variants that have improved binding afﬁnity. To achieve further improved anti~human 4-]BB dies, a 94/w light chain as described above was used and the 946] heavy chain was affinity matured. Provided in Table 2 below are the HCDR amino acid sequences for a reference 940! antibody heavy chain.

Table 2 - 9461 and 94K HCDR amino acid sequences SEQ ID NO 5 GYTFSSYW HCDR l SEQ ID NO: 6 INPGNGHT HCDR 2 SEQ ID NO: 7 ARSFTTARAFAY HCDR 3 HCDR 3.5 SEQ ID NO. 8. ARSFKTARAFAY variant 94K Improvement of a heavy chain using 94/w as a starting sequence was performed by similar methods as described for the 94G! light chain above. Particularly, to e a 94Gl heavy chain, amino acid residues were substituted with various amino acids at respective amino acid positions of HDRZ and/or HCDR3. In the case of the third CDR of the heavy chain (HCDR3, SEQ ID NO: 7), clones were produced with random substitution amino acid residues of 94/w HCDR3 by different amino acids were collected to e 12 position mixes. A mutant clone that ses the length of HCDR3 was also prepared. When the 5m amino acid residue of HCDR3 was substituted with a different amino acid, an afﬁnity increase was observed.

Subsequently, to detemtine how s mutations at the HCDR3.5 position impacted affinity of the 94/w antibody, 19 monoclonal antibodies were ed from a position mix in which the HCDR3.5 position of the 94/w antibody was randomly tuted. HCDR3.5 variant Fabs were expressed in Eco/i by adding lPTG (e.g., to a concentration of 1 mM) and ELISA was performed using a Fab antibody t in a atant. Sequencing identified that when threonine was substituted with lysine at HCDR3.5 (5"1 position) position (SEQ ID NO: 8 - ARSFKTARAFAY), the highest affinity was shown, and the resulting product was called 94K/w.

In the case of the second CDR of the heavy chain (HCDRZ), a position mix was prepared by random substitution of each of 9 amino acids of a 94G] HCDR2 (SEQ ID NO: 6) for ELISA. The ELISA results showed that when amino acid residues at 2"“, 5'“ and 6'h positions were changed, the afﬁnity increased. From each of the 94/w HCDR2.2, HCDR2.5 and HCDR2.6 on mixes, 22, I9, and 36 monoclonal antibodies were isolated, respectively, and the binding activity of each clone with respect to 4-1BB was analyzed depending on an Fab expression level In the case of HCDR2.5, an ELISA value was relatively higher than those when asparagine was substituted with valine (V), glycine (G), or proline (P). In addition, according to sequencing data for antibody heavy chains, there was a risk of deamination at the 5lll amino acid, asparagine (N). ofHCDR2 (SEQ ID NO: 6), and variant HCDR2 sequences were also ed with substitutions at this residue with each of glutamine (Q), glutamic acid (E), and serine (S).

DNAs of 9401 structural variants with mutations in HCDR3 and/or HDRZ of the heavy chain prepared as described above, were ampliﬁed by PCR using a three—base sequence NNS, ligated to the heavy chain position of a vector having a constant domain of the 94/w light chain, and then transformed into E. 0011' TO] by the method used in improvement of the light chain as described above. 1. 4 Optimization nized anti-human 4—!BB antibody heavy chain/i‘amework regions Heavy chain variants were also produced with optimized framework sequences.

For example, heavy chain framework ] (FRI) regions were ed where the heavy chain FR] (SEQ ID NO: 16) was modified so that the 5'" amino acid, glutamine (Q), was substituted with valine (V). ary FRI regions are ed in Table 3 below.

Table 3 — 94G] heavy chain FRI and ions thereof SEQ ID N0: 16 QVQLQQSGAEVKKPGASVKLSCKAS SEQ ID NO: l7 QVQLVQSGAEVKKPGASVKLSCKAS FRI Gln 5 Val |0232] Also, framework 3 (FR3) regions were produced where the heavy chain FR3 (SEQ ID NO: 18) was modiﬁed as such: the 10‘11 amino acid, e (A), and/or the 33rd amino acid, serine (S), which were murine sequences, were substituted with valine (V) and threonine (T), respectively. Exemplary FR3 regions are provided in Table 4 below.

Table 4 — 94Gl heavy chain FR3 and variations thereof SEQ ID NO: 18 NYNEKFKSRATMTRDTSTSTAYMELSSLRSED 94Gl FR3 SAVYYC SEQ ID N0: 19 NYNEKFKSRVTMTRDTSTSTAYMELSSLRSED FR3 Ala 10 Val SAVYYC SEQ ID NO: 20 NYNEKFKSRVTMTRDTSTSTAYMELSSLRSED FR3 Ala l0 Val; TAVYYC FR3 Ser 33 Thr 1.5 Preparation nized anti-human 4-]BB variable regions andﬂi/l-lenglh antibodies Anti-human 4-lBB antibody variable regions were produced that include various combinations of the above described heavy chain and light chain CDRs and framework regions.

For example, a Fab-type w antibody was produced with the 5lh amino acid, threonine, at CDR3 of a heavy chain was substituted with lysine (K), and the 10‘“ amino acid of heavy chain FR3, alanine, and the 33rd amino acid of heavy chain FR3, serine, were substituted with valine (V) and 'threonine (T), respectively to produce heavy chain and light chain variable region sequence that are or include SEQ ID NO: 30 and SEQ ID NO: 34, respectively. In addition, 94KVT heavy chain variants were produced where the 5“1 amino acid of HCDR2 (SEQ ID NO: 6), asparagine (N), was substituted with glutamine (Q) glutamic acid (E) or serine (S). ary heavy chain and light chain variable domain sequences are provided in Table 5 below (CDR ces underlined).

Table 5 — Exemplary humanized anti—human 4-1BB antibody le domains Antibody Light chain variable domain Heavy chain variable domain SPAFLSVTPGEKVTIT QVQLQQSGAEVKKPGASVKLS CRASQTISDYLHWYQQKPDQ CKASGYTFSSYWMHWVRQAP APKLLIKYASQSISGIPSRFSGS GQGLEWIGEINPGNGHTNYNEK TFTISSLEAEDAATYY FKSRATMTRDTSTSTAYMELSS CQDGHSFPPTFGQGTKLEK LRSEDSAVYYCARSFTTARAFA (SEQ ID NO: 9) XWGQGTLVTVSS (SEQ ID NO: ll) DIVMTQSPAFLSVTPGEKVTIT QVQLQQSGAEVKKPGASVKLS CRASQTISDYLHWYQQKPDQ CKASGYTFSSYWMHWVRQAP APKLLIKXLLSQSISGIPSRFSGS GQGLEWIGELWTNYNEK TFTISSLEAEDAATYY FKSRATMTRDTSTSTAYMELSS CQDGHSWPPTFGQGTKLEIK LRSEDSAWYCARSFTTARAFA (SEQ ID NO: l0) XWGQGTLVTVSS (SEQ ID NO: 11) SPAFLSVTPGEKVTIT QVQLQQSGAEVKKPGASVKLS CRASQTISDYLHWYQQKPDQ CKASGYTFSSYWMHWVRQAP APKLLIKmQSISGIPSRFSGS GQGLEWIGEINPGNGHTNYNEK GSGTDFTFTISSLEAEDAATYY FKSRATMTRDTSTSTAYMELSS CQDGHSWPPTFGQGTKLEIK AVYYCARSFKTARAFA (SEQ ID NO: 10) XWGQGTLVTVSS (SEQ ID NO: 12) DIVMTQSPAFLSVTPGEKVTIT QVQLVQSGAEVKKPGASVKLS ISDYLHWYQQKPDQ CKASGYTFSSYWMHWVRQAP APKLLIKXASQSISGIPSRFSGS GQGLEWIGEWNYNEK GSGTDFTFTISSLEAEDAATYY FKSRVTMTRDTSTSTAYMELSS CQDGHSWPPTFGQGTKLEIK LRSEDSAVYYCARSFKTARAFA (SEQ ID NO: 10) XWGQGTLVTVSS (SEQ ID NO: 13) 94KVT/w, DIVMTQSPAFLSVTPGEKVTIT QVQLVQSGAEVKKPGASVKLS CRASQTISDYLHWYQQKPDQ CKASGYTFSSYWMHWVRQAP APKLLIKY_ASQSISGIPSRFSGS GQGLEWIGEINPGNGHTNYNEK GSGTDFTFTISSLEAEDAATYY FKSRVTMTRDTSTSTAYMELSS CQDGHSWPPTFGQGTKLEIK LRSEDTAVYYCARSFKTARAFA (SEQ ID NO: 10) XWGQGTLVTVSS (SEQ ID NO: l4) For conversion to a ength anti-human 4-lBB antibodies (whole [g type), an Fc domain was ted to the respective Fab. For example, a 94K/w Fab composed of a heavy chain in which threonine is tuted with lysine at HCDR3.5 and a light chain with 94/w variant in which the 6'“ amino acid of LCDR3 is tuted with hyptophan (W), and respective regions extended from CH2 and CH3 domains and a sequence of human IgG1 were ampliﬁed by PCR to p and subjected to splice PCR to produce full [gG DNA, and then the resulting DNA was cloned in a mammalian expression vector. Full length antibodies for other humanized anti-human 4—IBB antibodies described herein were produced in a similar manner.

Exemplary immunoglobulin constant region sequences are provided in Table 6 below.

Table 6 — Exemplary immunoglobulin constant domains SEQ ID NO: 21 RTVAAPSVFIFPPSDEQLKSGTASWCLLNNF K constant YPREAKVQWKVDNALQSGNSQESVTEQDSK domain DSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC SEQ ID NO: 22 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY lgGl VSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID NO: 23 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY IgGl t VSWNSGALTSGVHTFPAVLQSSGLYS (L234; L235; LSSWTVPSSSLGTQTYICNVNl-IKPSNTKVDK [(3 22) KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN EVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCAVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLD LYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK As used herein a full length 94KVT/w antibody includes an IgGl sequence, such as that of SEQ ID NO: 22. Additionally, a full length antibody, referred to herein as EU 101, was produced that includes 94KVT/w variable domains descn'be above (SEQ ID N05: 10 and 14, for light chain and heavy chain variable domains, respectively), with a variant IgGl constant domain that includes 3 mutations: L234, L235, and K322 (SEQ ID NO: 23). Thus, example provides a number of ary humanized anti-human 4- l BB antibodies and antibody fragments that have been engineered to potentially enhance antigen binding afﬁnity. These exemplary antibodies and fragments are characterized in the following examples.

Example 2 — Characterization of binding of humanized anti—human 4-lBB antibodies 2. I DelermiI-Iing binding epilope Qfami-human 4-IBB all/ibodies The present disclosure encompasses a recognition that humanized anti-human 4- lBB antibodies ed herein may be useful for 4-1 BB co—stimulation. Therapeutic applications of dies of the present disclosure may include promoting anti-cancer immunity and/or anti-viral immunity. r, for clinical applications, it is important to identify which part of human 4-IBB is recognized by and/or reacts with an anti-humanized 4-lBB antibody (i.e., a binding epitope). 4- [BB antibodies that recognize different epitopes of 4-1 BB molecule have identiﬁed, and these antibodies can have been shown to have ent clinical effects. (See, e.g., Kwon et al. Eur. J. genetics (2002) 29: 449-452, herein orated by reference in its ty). Epitope mapping encompasses methods for identifying a molecular determinant of antibody-antigen recognition. This example describes epitope mapping of an exemplary anti- human 4-1BB antibody as engineered in Example 1 above. cally, this example assesses the binding epitope of a humanized anti-human 4-lBB antibody with 94KVT/w variable domains, EU101.

A human 4-lBB antigen for investigating an e of the humanized 4-lBB antibody is derived from a cDNA library ctured from human peripheral blood lymphocytes that was generated by at least some of the ors of the present application (See, e.g., Kwon et al. Cellular Immunology (1996) 169: 91-98; Immunol. Lett. (1995) 45: 67-73; and Korean Patent No. 10-0500286, each of which is orated herein by reference). cDNA encoding an extracellular domain (ECD) of the obtained human homologue of 4-1 BB cDNA (hereinafter, referred to as H4-1BB) was selected, fused with GST, and then ed into a vector (pGEX-6T) to express. A cell line producing a GST1BB fusion polypeptide as used herein, was deposited as part of the disclosure for Korean Patent No. 10-0500286, Accession No: KCTC 09SZBP. A full length sequence of human 4~lBB is provided as SEQ ID NO: 44, below.

The extracellular domain of human 4-lBB corresponds to amino acids 1 to 167 of the full length H4-1BB sequence.

SEQ ID NO: 44 - Full length human 4-] BB ce MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPN SFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSM CEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVN GTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLF LLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG |024Z| To ine an epitope of 4-lBB recognized by humanized anti-human 4-] BB antibodies of the present disclosure, constructs were generated with fragments of a 4- 1 BB ellular domain of various sizes (e.g., R1, R2, R3), fused to GST, and replicated. A schematic of GSTIBB polypeptides as used in the present example is provided in . and exemplary primer sequences used herein for generating different 4- [BB extracellular domain constructs are ed in Table 7 below. Individual recombinant GST-HIBB constructs were cultured with 1 mM IPTG and produced in E. coli BL2lDX5a cells, and the fusion polypeptides were purified using a glutathione-agarose column.

Table 7 — Exemplary primers used to generate human 4- 1 BB extracellular domain frauments useful for epitope mapping -5Fonward Reverse GGATCCACAAGATCATTGCA TTGAGCTCGAGCCTGGTCCTGAAA G-3’ (SEQ ID NO. 24) ACA-3(SEQ ID NO. 25) 57- S,- CGCGTGGATCCAAGGAGTGTTCCTC TCGAGACGTTTCTGATCG CA~3’ (SEQ ID NO. 26) TTA—3’ (SEQ ID NO. 27) ’- 5’— CGCGTGGATCCGGCATCTGTCGACC TTGAGCTCGAGGATCTGCGGAGAG CT-3’ (SEQ ID NO. 28) TGT-3‘ (SEQ ID NO. 29) ’- 5’- GGATCCACAAGATCATTGCA CTCGAGGCATATGTCACAG G-3’ (SEQ ID NO. 30) GT-3’ (SEQ ID NO. 3 l) ’- 5’— GGATCCACAAGATCATTGCA CTCGAGGCTGGAGAAACT G-3’ (SEQ ID NO. 32) AT-3’ (SEQ ID NO. 33) ’- 5’- GGATCCTGCCCAGCTGGTA TTGAGCTCGAGCCTGGTCCTGAAA C-3’ (SEQ ID NO. 34) ACA-3‘ (SEQ ID NO. 35) 52 5’- GGATCCAGGAATCAGATTTG TCGAGCCTGGTCCTGAAA C-3’ (SEQ ID NO. 36) ACA-3’ (SEQ ID NO. 37) ’- 5’- ACAAGATCATTGCA CTCGAGGCAAATCTGATTC G-3’ (SEQ ID NO. 38) CT-3‘ (SEQ ID NO. 39) ,- 55- GGATCCACAAGATCATTGCA CTCGAGTGGAGGACAGGGA G-3’ (SEQ [D NO. 40) CT-3’ (SEQ ID NO. 41) Puriﬁed protein samples were obtained from transformed bacterial cells by a lysis buffer (e.g., 10 mM Tris-HCI — pH 7.4, 50 mM NaCl, 5mM EDTA, 30 mM NaF, 0.1 mM Na3VO4, 1% Triton X-100, 0.5% t P—40, 1 mM PMSF, and protease inhibitor mixture).

Approximately 20 pg of each fusion polypeptide sample was diluted in a 4X SDS sample buffer, subjected to electrophoresis on SDS-PAGE gels, and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). On the cellulose membranes, anti-human 4-] BB mAb was reacted with anti-mouse lgG adish peroxide (HRP). Binding antibodies were recognized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Little Chalfont, UK).

As described above and shown in , when each of three non-overlapping H4-lBB ECD fragment-GST fusion polypeptides, R1, R2, and R3, were treated with GST- binding, respectively. It was determined that an exemplary humanized antilBB dy encompassed by the present disclosure (EUl 01) binds to an N-terminal fragment construct (Rl) fusion uct of approximately 32 kDa (amino acids 1 to 55 of 4-IBB) by western blotting.

Moreover, this binding was speciﬁc, as no binding was observed with either of the R2 or R3 fusion constructs. See .

Furthermore, to ine the minimal binding site of the humanized antilBB antibody, an R1 extracellular domain fragment was further divided into 6 smaller fragments: R1,] (amino acids 1 to 45 of4—lBB), R12 (amino acids 1 to 35 B), Rl.3 (amino acids H to 55 of4—lBB), R14 (amino acids 2] to 55 of4-lBB), R1 .5 (amino acids 1 to 25 of4—lBB), and R] .6 (amino acids 1 to 30 of 4-1BB) polypeptide fragments, as depicted in , and WO 2018/]27787 PCTllBZOIS/(Ilﬂl043 fused to GST (Glutathione S-Transferase, 27 kDa). ary primer pairs used for the tion of these constructs are provided in Table 7 above. Fusion ptide constructs were produced in E. coli BLZ] cells with IPTG induction (e.g., lmM IPTG) and bacterial whole cell extract was ed by 12% SDS-PAGE. As shown in , SDS-PAGE conﬁrmed that individual 4-IBB fusion polypeptides are well expressed.

SDS-PAGE was transferred to a nitrocellulose membrane and blotting was performed using an exemplary anti-human 4~lBB antibody. EUlOl. As shown in , it was ed that a ce of amino acids 10 to 30 of the extracellular domain of H4- l BB is signiﬁcant for binding an exemplary humanized antilBB antibody. This analysis indicates that an exemplary anti-human 4-lBB dy of the present sure (EUlOl) binds to an epitope of human 4—1BB whose sequence is or includes CPAGTFCDNNRNQICSPCPP (SEQ ID NO: l5). It was also conﬁrmed that a sequence including amino acids 35 to 50 of the 4-lBB extracellular domain is not signiﬁcant for binding an exemplary humanized antibody described herein (FIG. ZB). 2.2 Assessing binding qfﬁnily ofexemplary humanized anti-human 4—18/3 antibodies to J—IBB antigen Binding ability of exemplary anti-human 4-1 BB antibodies To examine the binding ability of exemplary humanized anti-human 4- 1 BB antibodies described in Example I to a human 4-lBB antigen (H4—lBB). ELISA was performed.

E. ooh-expressed recombinant human 4-lBB was used for antigen.

A murine BBK-4 antibody, a reference 940] humanized antibody, and exemplary engineered antibodies 94K, 94KV, 94KVT and EUlOl as described in Example 1 were each d on 96 well plates coated with histidine—tagged 4- 1 BB extracellular domain recombinant protein (H4-1BB). Exemplary ELISA afﬁnity analysis employed a total volume of mo pl at a concentration of 10 ug/ml, and the reaction was allowed to proceed at room temperature for 1 hour. Horseradish peroxidase (I-IRP)-labeled anti—human IgG and lgG-HRP, as appropriate, recognizing an antibody was treated thereto, and allow to react at room temperature for 40 minutes. After washing, treatment with an ABTS solution (Sigma-Aldrich), which is a substrate for a ng reaction, and the reaction to allow to d at room temperature for 30 minutes, and an absorbance at 450 nm in the coloring on was detected using an ELISA reader to analyze a binding activity of the antibodies. Results are shown in As shown in as antibody concentration ses, binding between each antibody and 4- | BB antigen (H4-1BB) is improved This data conﬁrms that antibodies encompassed by the t sure speciﬁcally bind to 4-1 BB.

Binding of exemplary anti-human 4-1BB antibodies to cell-expressed antigen The ability of exemplary humanized anti-human 4- [BB antibodies to bind a human 4- I BB antigen (H4-l BB) in a cellular t was assessed. Iurkat 8-l cells were genetically engineered for overexpressing 4-IBB. ary engineered antibodies 94K, 94KV, 94KVT and EU l 0] as described in Example I, along with that of a murine BBK-4 antibody, and a reference 940] humanized antibody were each assessed for binding to Jurkat 8~l cells using an anti-mIgG-HRP or IgG-HRP secondary antibody, as appropriate, and analyzed by FAC S.

As shown in each of the antibodies were able to effectively bind 4-IBB expressed by Jurkat 8-I cells and the afﬁnity of 94KVT and EUIOI was higher than BBK-4 and 9401, In vitro binding affinity of exemplary anti-human 4-lBB antibodies to antigen [025” In vitro binding affinity of exemplary engineered antibody EUIOI as described in Example I, along with that of a reference 940] humanized antibody were each determined by Biacore analysis. Anti-human IgG was immobilized on a CMS chip, and coupled to the Fab antibodies prepared above by ﬂowing over the chip, and ultimately reacted with a human 4-lBB antigen (H4—IBB) to measure the g activity between the antibody and the antigen re3000, sensor chip CMS). Afﬁnity measurement results are shown in Ka (l/Ms) and Kd (l/s) values ent how fast an antibody associates with and iates from an antigen, respectively. A iation constant (K9) is obtain by dividing Kd by Ka (Kd/Ka= KB).

As a dissociation constant decreases, it can be reted that dissociation occurs at a lower concentration and that affinity is sing. As shown in the exemplary engineered anti-human 4—lBB antibody had improved binding afﬁnity relative to a reference 94Gl.

Exemplag anti-human 4-IBB antibodies recognize 4-1BB expressed by activated CD8‘ T cells CD8‘ T cells were isolated from human PBMCs and activated with lug/ml anti— CD3 antibody —for 2 day. The ability of exemplary humanized anti-human 4- [BB antibodies (94K, 94KV, 94KVT and EUIOI) described in Example 1 to detect a 4-1BB on the surface of activated CD8’ T cells was assessed relative to an exemplary commercially available anti [BB antibody (4-lBB-PE). Also shown is detection with a BBK-4 a murine anti—human 4-lBB antibody and a 94Gl reference humanized antibody. Treatment with 4—] BB antibodies was at a concentration of 25 ng/ml.

Exemplary antibodies were detected with an anti-mIgG~Dylight488 or anti-h1g6- Dylight488 as appropriate, and analyzed by FAC S. s are shown in While a reference 946] antibody ed 4-IBB on 17.93 "/0 of CD8- T cells, each ofa 94KVT and EUlOl antibody showed robust detect of 25.3% and 28.33%, respectively. Demonstrating that exemplary antibodies 94KVT and EUlOIboth had improved binding properties over BBK—4 and 9401. Thus, humanized variant antibodies of the present disclosure have superior binding to activated T cells in vilro.

Example 3 - Analysis of in vitro cy of humanized anti-human 4-IBB antibodies AntiIBB antibodies have usly been demonstrated to provide signal stimulation to a co-stimulation molecule expressed in ted CD8. T cells, 4- I BE, to activate the CD8” T cells, induce proliferation and increase TH l-type cytokine expression. In this example, activity of ary humanized uman 4-lBB antibodies described in Example I to induce proliferation of CD8* T cells and THl-type cytokine sion was examined. 3.] Exemplary uman 4—lBB antibodies induce cell pro/(fanatic): QfCD8+ Toe/ls To assess proliferation ofCD8‘ T cells, cells were d with WST-l (water- soluble tetrazolium salt) is a cell proliferation reagent. WST-l—labeled CDST T cells were prepared and activated with 0.5 ug/ml of anti-CD3 antibody. The activated CD8’ T cells were treated with 1,0 [lg/ml of iso-type l antibody, mutine BBK-4 antibody, reference 946] antibody, and exemplary humanized anti-human 4-lBB antibodies (94K, 94KV, 94KVT and EUlOl) described in Example 1. Cells were analyzed using a MACS system and results are shown in ing to it was confirmed that exemplary humanized anti-human 4- 1 BB antibodies of the present disclosure induce cell proliferation of CD8‘ T cells. er, a degree of CD8’ T cell activation increases in an order of 946] < 94K/94KV < 94KVT / EU WI. 3. 2 Exen-iplary anti-human 4- 1BB antibodies slimy/ale ne secretion IFN-y is a representative cytokine ily secreted from a T lymphocyte or a natural killer cell (NK cell) and exhibiting eration and anti-viral activities. In on, [FN- y is a major activator for a hage, and particularly, a major cytokine distinguishing TH] cells from other types of cells. IFN—y secretion plays a major role in the activation of cytotoxic T cells, phagocytes and B cells. Consequently, efficiency of an anticancer agent can be evaluated with an increased amount of TH] inducing IFN-y. For this reason. measurement of secretion of LFN-y by speciﬁc stimulation may be an optimal standard which can be used as a quantitative criterion for a functional change of T cells.

CD8- T cells were isolated from human PBMCs and treated with 05 ug/ml of an anti-CD3 mAb antibody and then treated with either no antibody, or with 1.0 [1le of an anti IBB antibody: BBK-4, 940 I, 94K, 94KV, 94KVT and EUlO l. IFNy secretion was evaluated on days I, 3, and 5. Results are shown in As can be seen in IFNy secretion increased in all antilBB dy treated samples, and this increase correlated with duration of dy treatment. Treatment with 94KVT and EUIOI antibody reached a secretion level that was 13-fold higher than the control group as day 5. Accordingly, exemplary humanized antibodies 94KVT and EUIOI can both induce [FNy secretion more efficiently than 94G] reference antibody. 3. 3 Increase in lFN—y level according to ent afaclivaled (‘D-i"; Tee/ls or (‘1)8‘ Tee/ls with an exemplmy uman 4- 1BB antibody Blood was collected from three healthy donors, PBMCs obtained there from were isolated by Ficoll—plaque gradient centrifugation, and active T cells present in the PBMCs were rested in a RPMI- l 640+2°/o FBS medium for 24 hours. The rested PBMCs were treated with an iron beads-attached anti-CD4 antibody or anti-CD8 antibody, and CD4' cells or CD8‘ cells were isolated using an MACS magnetic separator. The isolated CD4- T cells or CD8' T cells were treated with a T cell activator, anti —CD3, to induce 4-IBB expression, and treated with EUlOl at different concentrations (0.5, 1.0, 2.5, and 5.0 ug/ml) for 3 days. After 3 days, a culture medium excluding the cells was obtained, and ﬂuorescence of human IFN—y in the culture medium was assessed by ELISA (ebioscience), and the result was compared with the standard curve provided in an IFN-y ELISA kit (.

As shown in expression levels of [FN-y in the CD4' T cells and CD8' T cells dose-dependently increased. Particularly, when 5.0 ug/ml of EUIOI was treated, compared with a 278% increase in the CD4' T cells, the expression level of IFN-y increased 612% in the CD8‘ T cells. According to the T-cell speciﬁc expression pattern of lFN-y involved in the conversion of the T cells into TH I, an exemplary anti-human 4-lBB antibody of the present disclosure, EU I 01, has sufﬁcient in vii/‘0 activity to suggest it may be ive for prevention and/or treatment of cancer. 3. -/ Measure/”en! ofAlX‘C and (‘1)(‘ aclivilies ofan exemplary (viii-human 4—IBB antibody ] An immune system recognizes and s virus-infected cells or cancer cells, and antibodies may be used to induce xicity mediated sis. For such an immune , two types of mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) may be used. In both cases, apoptosis may be mediated by an antibody binding to a target on a cell surface. That is, when an antibody has an ADCC ty, a cell ized by the antibody results in apoptosis mediated by a natural killer (NK) cell, and when an antibody has a CDC activity, g is mediated by a complement protein. Therefore, in the case of the development of an antagonistic antibody therapeutic, a degree of g cells ized by an antibody can be identiﬁed through analyses of the ADCC and CDC activities. However, a target for the humanized 4-lBB antibody disclosed in the t disclosure is T cells, not cancer cells. That is, in consideration of a mechanism for inducing activation of T cells by binding a 4-l BB dy as an agonistic antibody, an antibody that does not have the ADCC and CDC activities may be preferably for therapeutic uses.

In the present disclosure, for an ADCC assay, human PBMCs were isolated by Ficoll centrifugation using the same density difference. The PBMCS were incubated into RPM] (Thermo Fisher Scientific) and 10% PBS with lL-2 (lOOU/ml) for overnight cultured. Target cells (4-IBB expressing cell lines) were harvested, resuspended in a culture medium at I ml, and labeled with 5 uM CFSE at 37 °C for 5 min. Effector/Target cells of the present disclosure were washed in a ratio of IO: 1 counted and then dispensed. For analysis, an antibody of the present disclosure was prepared for a final concentration of 10 nM (1.5 rig/ml), and cultured on a plate at 37 °C for 4 hours. 5 ul of 7-AAD was added to each well and transferred to a FACS tube, and then the sample was analyzed by FAC S manufactured by BDFACScan. Frequencies of non- viable target cells (CFSE ' 7—AAD ') viable target cells (CFSE ’ ) were measured.

ADCC was assessed with a frequency of viable cells of the total cells (A).

A complement-dependent cytotoxicity (CDC) assay was ted similarly to the ADCC assay described above using FACS as a read-out value, with the above Target cells incubated with anti—4-IBB dies at ice for 30 min and then added the human supplemented serum at a ﬁnal concentration of 20 % at 37 °C for 30 minutes. ard, resulting samples were each transferred to a FACS tube, and assessed by FACS manufactured by BDFACScan (FIG. IOB). The results in A and B conﬁrm that an exemplary humanized 4- 18B antibody, EUlOl, has almost no ADCC and CDC effects. Therefore, it can be said that an exemplary EUlOl antibody of the present sure has beneficial ADCC and CDC properties for an agonist antibody, and is a good candidate for ancer treatment in viva.

Example 4 — Confirmation of in viva ncy of an exemplary humanized anti-human 4- 188 antibody The anti-human 4-] BB antibody, EU l 01, of the present disclosure showed a dose- ent effect in an in vim) example, and showed a considerably superior effect to a conventional antibody. This example is to check if the anti-human 4-1BB antibody, EU 10], is able to be used alone or in combination with a different composition to diagnose, prevent or treat cancer or tumor in viva, and to effectively inhibit the growth of tumor. 4.1 NOD-said11.2Rgammdm” mouse engrqftmem of human peripheral blood mononuclear cells and anti-tumor activity ofanti-hllmml 4—188 antibody Peripheral venous blood collected from a 4-type healthy donor was treated with heparin, and subjected to concentration-gradient centri fugation on -paque (GE Healthcare, Piscataway, NJ) to harvest PBMCs. The PBMCs were washed with an RPMI-1640 medium, and 3x106 of the cells were intraperitoneally injected into immnodeficient mice, that is, NSG mice (NODCg-Prkdcm" l/2r_ ”WI/82]; NOD-scid ILZry""", Jackson Laboratory). is of humanized mice was performed by ﬂow cytometty to check whether human T cells were present in the mouse blood collected by mouse orbital blood tion after weeks of the ment of human PBMCs. 7-week-old NSG mice (Jackson Laboratory, Barharbor, ME) were raised under a speciﬁc pathogen-free (SPF) environment.

Flow cytometry was performed to check ratios of CD4 and CD8 after the cells were stained with human blood cell markers such as an APC-cy7 ﬂuorescence- labeled CD45 antibody and a FITC fluorescence—labeled CD4 antibody, and a BVSlO cence-labeled CD8 antibody. After l blood collection from each mouse, human T cells from mouse blood samples were observed to check if a human immune system is engrafted into the mouse. Human tumor cells were prepared in an HLA type humanized mouse model and l x 107 cells were subcutaneously injected into the back of each mouse. When a tumor size reached 100 to 200 mm3, a preparation of exemplary anti-human 4-1BB antibody (EU 10]) was intravenously administered at 1.0 mg, 5.0 mg or 10.0 mg per 1 kg of body weight once every 5 days total 3 times. As a control, human IgG was used. Tumor volume (mm3) of each mouse was measured in every 3 days (). Results shown conﬁrm that tumor size in mice treated with an exemplary anti-human 4-] BB antibody (EUlOl) was reduced relative to mice treated with human lgG, and moreover that this ion was proportional to antibody concentration.

Particularly, tumor regression in a 5 mg/kg dy-administered group occurred rapidly.

Within a week after stration at a 5 mg/kg dose, tumor size settled in a zed mouse and tumor growth was eradicated. Therefore, an exemplary antibody EUlOl of the present disclosure shows an anticancer effect in viva.

Consequently, the above results show that an exemplary anti-human 4-lBB antibody (EUlOl) that speciﬁcally recognizes an epitope (SEQ ID NO: 15) of H4-IBB, but due to improved characteristics of this exemplary antibody, such as, for example, improved afﬁnity, this antibody shows superior effects in an in viva mouse model. Thus, the example suggests that an antibody assed by the present disclosure can be used as an anticancer agent at a lower dosage than reference antibody. 4. 2 Is Qfmhibiling IllmOI' growth with an exemplary anti—human 4—!BB antibody and an anti-PD-I agent Comparison of effects caused by individual treatment of an exemplary anti-human 4-lBB antibody (EU 1 01 i and an exemplary anti-PD-l agent after tumor injection to humanized mice Humanized mice were prepared by the same method described in Example 4.] above. To perform an experiment confirming an increase in anti-cancer effect according to doses of an exemplary anti-human 4-lBB antibody (EUIOI) and an ary D-l agent (Keytruda)(purchased from MSD, GER), l x 107 cells of a HLA—A-type matched human colorectal adenocarcinoma cell line, HT29, were subcutaneously injected into the previously~ prepared humanized mice. When the volume of the injected tumor reached 100 to ISO mm}, the mice were divided into a total 5 groups of three mice, and to compare the effect of EUlOl on tumor inhibition, each group of mice were treated with each of ﬁve three administration conditions (Control: IgG, Treated group 1: 5 mg/kg, and Treated group 2: 10 mg/kg) at 5 day intervals 5 3 times, and for anti-PD—l, the same procedures were carried out (). As a result of the experiment, in both cases of EUIOI and keytruda (anti-PDI), tumor volumes were dose-dependently reduced. However, in , 5 mg/kg of EUlOl did not have an influence on the tumor growth, but according to the ent with 5 mg/kg and 10 mg/kg of EUlOl, an anti-tumor activity was ependently ted. In addition. it was confirmed that EUlOl ted higher efficiency at a lower dose than keytruda (anti-PD-l ), and the tumor growth was tely blocked ularly by the treatment with 5 mg/kg of EUlOl.

Treatment of humanized mice with combination of EU 1 Oland an anti-PD-l agent after tumor injection Since co-inhibitory receptors (PD-l and CTLA-4) signals and a co-stimulation (CD137)T cell signal are differentiated for the same purpose of inhibiting tumor growth, ation of the two receptors can expect a synergyic effect (Chen er al., Cancer l. Res. (2015) 3: l49-160; Bartkowiak et 0]., From 011001. (20l5) 5: I 17, both of which are incorporated by reference herein). In addition, PDI imrnunotherapy showed a possibility of an anticancer treatment effect for some of cancer patient populations, but the administration of a low dose in combination therapy with a different anticancer agent may still be ed in more extensive patient population. To igate the anti—tumor effect caused by a combination y of an exemplary anti-human 4—lBB antibody (EUIOl) and an exemplary anti-PD-l agent (Keytruda), tumor-bearing humanized mice were treated with the combination therapy of EU 1 0| and Keytruda. Preparation of humanized mice was performed by the same method as described in Example 4.1 Eye bleeding was performed to identify humanized mice. Among the humanized mice, HT29, colon carcinoma were subcutaneously injected into HLA-A24 mice maintaining a normal condition at lxlO7 cells/mice. When a tumor size was 300 to 450 mm3, an experiment was performed as follows.

As known from this example, gh tumor growth was not d with individual injection at the minimum concentration or less (EUlOl: 2.5 mg/kg, Keytruda (manufactured from MSD, GER): 2.5 mg/kg), tumor was greatly regressed with combination y of EUIOI and Keytruda. This is the result showing that exemplary anti-human 4-lBB antibodies provided herein (e.g, EU] 01) are good candidates for combination therapy with different anticancer agents, including in combination with one or more immune checkpoint inhibitors ().

Analyses of T cell inﬁltrating cytes [TILs] in normal tissue and human colorectal adenocarcinoma tissue after individual and combination treatment of an exemplam anti-human 4-l BB antibody and an exemplam anti-PD-l agent [0273'] After individual administration of an exemplary anti-human 4-lBB antibody (EUlOl) and an exemplary D-l agent (Keytruda) (purchased from MSD, GER) and combination administration of EU] 01 and Keytruda to HTZQ-implanted zed mice, on the day when the effect analysis is ated, all groups were ted to separate tumor and blood. After the ted tumor was treated with collagenase [V at 37 °C for 30 minutes, cells in the tumor tissue were dissociated by a mechanical method and then washed with leBS. PBMCs were ted from the separated blood by Ficoll gradient centrifugation, and separated tumor cells and PBMCs were subjected to the following experiment. Red blood cells (RBCs) were d from washed cells using RBC lysis buffer and then washed with leBS. Tangled cell debris was removed from the washed cells using a 40-um nylon cell strainer to create a single cell state, and the single cells were washed with leBS, followed by counting T cells separated from each group using a cell counter.

The separate T cells were stained with human blood cell markers such as a CD45 antibody (fluorescent APC-cy7 labeled), a ﬂuorescent FITC-labeled human CD4 antibody and a ﬂuorescent BVSIO-labeled human CD8 antibody, and then subjected to FACS assay. The FACS assay was carried out based on a ratio (9/0) of CD4 and CD8 cell groups, which were gated from the CD45 group (A).

Particularly, to identify a Treg group among the separated T cells, the surfaces of cells were d with human blood cell s such as a CD45 antibody scent APC- cy7 labeled), a human cent FITC-labeled CD4 antibody and a human ﬂuorescent PE-cyS- labeled CD25 antibody, and ellular and intranuclear staining with a cell transcription factor Foxp3 (human fluorescent APC-labeled Foxp3 antibody) were performed using a Foxp3/ Transcription Factor Staining Buffer Set kit (ebioscience). In the PAC S assay, a CD45 group was separated to gating RI , a CD4°CD25high group was separated to gating R2, and a ratio (°/o) of a Foxp3high group was measured in the RI and R2 groups. To identify IFN-y'CD8' T cells in the separated cells, the cell es were stained with the blood cell markers such as the ﬂuorescent APC—cy7-labeled human CD45 antibody and the ﬂuorescent BVSlO—labeled human CD8 antibody, ﬁxed with 2% PFA, and reacted with a 0.5% saponin solution and a ﬂuorescent PE— cy7-labeled human IFN-y antibody. Afterward, cytokine IFN-y’ cells in the CD8 T cell group were measured by FACS assay. The cells were identiﬁed in a ratio by the same method as described above and a proportional ratio of the CD8‘IFN-y’ ratio and the Treg ratio was calculated, shown in 8.

According to the result of this embodiment, other than the individual administration, the combination administration of EUlOl and Keytruda greatly increased inﬁltration of the combination of tumor tissue and a T lymphocyte. Further more speciﬁc results of the combination treatment are as follows. When the ation treatment was performed on PBMCs in the y humanized mouse as a control, the number of lymphocytes sed approximately 3 times, and the infiltrated cytes per 1 g of tumor increased 76 times in tumor tissue. This means that most of tumor-speciﬁc lymphocytes were activated and recruited to tumor tissue to kill target cells. ularly, when PBMCs in the combination y group were measured, as shown in A, the CD4‘ T cells do not highly increased, but cytotoxic CD8' T cells were increased approximately 5 times. Moreover, the combination therapy group showed a lOO-fold increase in CD8~ Tcell count per 1 g of tumor tissue. In addition, as a result, a ratio of CD8' T cells secreting IFN-y and regulatory T cells was also greatly increased (B). That is, it can be said that the combination treatment of EU 1 Ol and anti-PD-I agent gives a sharp increase of effector T cells and thus tumor inhibition is effectively ied. es of IFN-x in serum or tumor ﬂuid obtained from human ctal adenocarcinoma tissue after individual and combination treatment with an ary anti-human 4-1BB antibody gEUlOl') and an exemplary anti-PD-l agent (Keytruda) After individual administration and combination administration of an exemplary anti-human 4-l BB antibody (EU [0 l) and an exemplary anti-PD-l agent (Keytruda) to I-IT29- implanted humanized mice. On the day when effect. analyses were ated, all groups were dissected to separate tumor and blood. In tumor dissection to separate a tumor ﬂuid present in the separated tumor, 300 pl of 1x PBS was injected into the upper n of a tumor membrane using a Ice—syringe, and a ﬂowing solution is taken from the lower n of the tumor membrane using an insulin syringe. In addition, in dissociation of the tumor tissue, the taken solution was added to dissociate the tumor tissue, and then stored. In addition, as a serum, the serum stored when PBMCs were separated from blood by Ficoll gradient centrifugation. The stored serum and a tumor fluid were dissolved and ﬁltered using a 0.22 pm ﬂiter unit (manufacturer: coming). 10 pl of serum was used for each group, and IOO ul of the tumor ﬂuid was used to measure human IFN-y and human TGF-B using a human [FN-y ELISA SET- Go kit (eBioscience) and a Human TGF beta 1 ELISA Ready-SET-Go kit (eBioscience).

Results were analyzed by comparing the standard curve provided in each ELISA kit.

As a result, compared to the individual administration of EUIOIand Keytruda, in the combination stration, the tration of interferon in serum of the tumor group was the highest. Since a EUIOI mechanism can be explained with a correlation between LPN-y and an anti-tumor effect, expression levels of IFN-y and TGF-B in serum of a healthy donor and serum of a tumor group, to which the combination therapy had been applied, were evaluated.

According to the material of the example on the serum of the healthy donor, in the combination therapy group shown in A, IFN-y was increased approxiamtely 16 times, but a cytokine secreted from Treg cells, TGF-B, was decreased approximatley 65%. In addition, in 3, the IFN-y concentration caused by the combination administration in the tumor ﬂuid was considerably higher (approximately 213-fold) than that in the control. As a result of the examples, due to EUIOI, particularly, compared to the control group, the combination group showed sharp increases in IFN-y secretion. Therefore, it can be conﬁrmed that the anti-cancer effect caused by an improved anti-humanized 4-l BB antibody of the present disclosure gives effective tumor-inﬁltrati on of effector T cells directly d to apoptosis of cancer cells, and a erably speciﬁc effect in the tumor tissue, compared to the non-treated group. In other words, in the present disclosure, it was confirmed that EUlOl, as an anti-cancer agent, has the optimal conditions for sis of cancer cells. Conventional] y, in cancer patients, anti-cancer cytokine and anti—cancer cellular immunity were considerably reduced, but it can be expected in the present disclosure that EU l O] induces the increases in anti—cancer cytokine and anti-cancer cellular ty, resulting in a considerable therapeutic effect.

Thus, an exemplary anti-human 4-1 BB antibody EUIOI exhibits an anti-tumor effect mediated by the high expression of IFN-y, and such an effect is dose-dependently exhibited, as such, an IFN-y concentration in a serum of a cancer t can be used as a biomarker to diagnose and estimate tumor. Therefore, according to effective treatment of cancer or tumor through the combination treatment of EUIOl and anti-PD-l and progmosis through the measurement of an lFN-y concentration. it is expected to perform more effective treatment with respect to each patient.

Example 5 — Separation and massive proliferation of CD8+T cells ex vivo using an exemplary humanized anti-human 4-lBB antibody The inventors used 4-lBB expression in antigen-speciﬁcally activated CD8+ T cells in isolation and puriﬁcation of 4-lBB'CD8° T cells speciﬁc to various antigens using an -lBB antibody (Korean Patent No. 104503341). A subsequent experiment was performed to examine if the EU l 01 antibody developed herein is also used for isolation and mass- proliferation of antigen-speciﬁc CD8‘ T cells. [028 l[ uction of PBMCs from peripheral blood of a cancer patient was performed as described in Example 4. 1. However, in this example, cancer antigen-speciﬁc undifferentiated T cells may be obtained by the method described in Korean Patent Application No. 10 0165224, ﬁled by the inventors. In this e, for effective separation of 4-1 BB‘CD8’T cells and roduction of the 4-1BB'CD8'T cells with high purity, a g method using an anthhuman 4—lBB antibody (EUIOI) was used. 10 ug/ml of the uman 4-lBB antibody (EU lOI) antibody diluted in PBS was added to a 10 ml ﬂask, and then stored at 4 °C for 20 to 24 W0 27787 PCT/lBZOIS/(lllll043 hours. After storage, a supernatant containing the dy was removed, and without washing, a solution ot'BSA dissolved at 2.5% in PBS was added to cell pellets in the 10 ml ﬂask and then stored at 4 °C for 20 to 24 hours Afterward, the BSA solution was removed, and each flask was washed twice with 15 ml of PBS. The previously-prepared cells were ded in an X-VIVO medium, added to a EUIOI antibody-coated ﬂask, and then incubated at 37 °C in a C02 incubator for 1 hour. After incubation, a supernatant was removed, and cell pellets were washed twice with 10 ml of RPMI] 640 medium to remove non-speciﬁcally binding cells. 1% of self serum and a IOOO IU/ml lLcontaining X-VIVO 10 medium were added to the ﬂask, followed by ing for 14 days. In the example, some cells were harvested and then stained to measure the purity and phenotypes of the isolated cells. As shown in FIGS. 16A and 168, it was conﬁrmed that, before g with the 94 kvt antibody, a ratio of antigen-specific 4-lBB-CD8' T cells increased 43.2% (CD8' T cell ratio: 586%), and after g with the EUIOl antibody, a ratio of antigen-speciﬁc pCMV'CD8' T cells increased 60.0% (CD8- T cell ratio: 79.3%). This means that the antigen-specific 4-lBB’CD8‘ T cells can be isolated with high purity using a EU l0]. Antigen-specific 4-1 BB’CDS' T cells isolated as described above may be easily mass- produced by the method described in Korean Patent Application No, lO-20l6-0165224 ﬁled by the inventors.

From the above description, it will be understood by those of ordinary skill in the art that the present invention can be realized in different specific forms without ng the technical idea or essential characteristics of the present invention. However, there is no intention to limit the present invention to the speciﬁc exemplary embodiments, and it should be understood that all modifications or modified forms deduced from the meaning and range of the following claims and equivalents thereof are included in the scope of the present disclosure, rather than the detailed description. uman 4—] BB antibodies encompassed by the present disclosure demonstrated a number ofbeneﬁcial properties, such as, for example, superior affinity to a nce antibody, and/or can be used alone or in combination with another anticancer agent to diagnose, prevent or treat cancer or tumor, or used to inhibit the growth of cancer.

Above, the present invention has been described with reference to examples, but it can be understood by those of ry skill in the art that the present ion may be changed and modiﬁed in s forms without departing from the spirit and scope of the present invention, which is described in the accompanying claims.

E UIVALENTS Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims.

Claims

1. A nucleic acid molecule encoding an anti1BB antibody or antigen-binding fragment comprising: (a) a heavy chain CDR1 comprising a sequence of SEQ ID NO: 5, a heavy chain CDR2 comprising a sequence of SEQ ID NO: 6 and a heavy chain CDR3 comprising a sequence of SEQ ID NO: 7 or 8; and (b) a light chain CDR1 comprising a sequence of SEQ ID NO: 1, a light chain CDR2 comprising a ce of SEQ ID NO: 2 and a light chain CDR3 comprising a sequence of SEQ ID NO: 4; (c) a light chain variable domain comprising a ce at least 98% identical to a sequence of SEQ ID NO: 10; and (d) a heavy chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 14, wherein the heavy chain variable domain ses a valine at position 5, a valine at position 68, a threonine at position 91, and a lysine at position 101.

2. A recombinant vector comprising the nucleic acid le of claim 1.

3. A host cell comprising the recombinant vector of claim 2 and/or the c acid molecule of claim 1.

4. The host cell of claim 3, wherein the host cell is selected from a bacterial, yeast, insect or mammalian cell.

5. The host cell of claim 4, wherein the host cell is selected from the group consisting of , P.pastoris, Sf9, COS, HEK293, CHO, and a ian lymphocyte.

6. Use of an anti1BB antibody or antigen-binding fragment comprising: (a) a heavy chain CDR1 comprising a sequence of SEQ ID NO: 5, a heavy chain CDR2 comprising a sequence of SEQ ID NO: 6 and a heavy chain CDR3 comprising a sequence of SEQ ID NO: 7 or 8; and (b) a light chain CDR1 comprising a sequence of SEQ ID NO: 1, a light chain CDR2 comprising a sequence of SEQ ID NO: 2 and a light chain CDR3 comprising a sequence of SEQ ID NO: 4; (c) a light chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 10; and (d) a heavy chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 14, n the heavy chain variable domain comprises a valine at position 5, a valine at position 68, a threonine at position 91, and a lysine at position 101, a nucleic acid of claim 1, or a recombinant vector of claim 2, in the manufacture of a medicament for the treatment of .

7. Use of an -1BB antibody or antigen-binding fragment comprising: (a) a heavy chain CDR1 comprising a sequence of SEQ ID NO: 5, a heavy chain CDR2 comprising a ce of SEQ ID NO: 6 and a heavy chain CDR3 comprising a sequence of SEQ ID NO: 7 or 8; and (b) a light chain CDR1 comprising a sequence of SEQ ID NO: 1, a light chain CDR2 comprising a sequence of SEQ ID NO: 2 and a light chain CDR3 sing a sequence of SEQ ID NO: 4; (c) a light chain le domain comprising a ce at least 98% identical to a sequence of SEQ ID NO: 10; and (d) a heavy chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 14, wherein the heavy chain variable domain comprises a valine at position 5, a valine at position 68, a threonine at position 91, and a lysine at position 101, a nucleic acid of claim 1, or a recombinant vector of claim 2, in the manufacture of a medicament for inducing an immune response or increasing the activity of an immune cell.

8. The medicament of claim 6, where in the cancer is selected from a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall r cancer, gastrointestinal cancer, head and neck cancer, hematological , laryngeal cancer, liver cancer, lung cancer, ma, ma, mesothelioma, ovarian , primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer.

9. The medicament of claim 6, wherein the subject has been administered or will be administered one or more additional anticancer ies selected from ionizing radiation, a chemotherapeutic agent, an antibody agent, and a cell-based therapy, such that the subject receives treatment with both.

10. The medicament of claim 9, wherein the one or more additional anticancer therapies comprise an immune checkpoint inhibitor, IL-12, , an anti-CD4 agent, cisplatin, uracil, doxorubicin, irinotecan, paclitaxel, or cyclophosphamide.

11. The medicament of claim 10, wherein the t has been administered or will be administered a composition comprising an immune checkpoint inhibitor, such that the subject receives treatment with both.

12. The medicament of claim 11, wherein the immune checkpoint inhibitor is an agent that inhibits PD-1 ing.

13. The medicament of claim 12, wherein the agent that inhibits PD-1 signaling is a PD-1 antibody.

14. A method for increasing secretion of IFN-γ by a cell in vitro, the method comprising: ting the cell with an anti1BB dy or antigen-binding fragment comprising: (a) a heavy chain CDR1 comprising a sequence of SEQ ID NO: 5, a heavy chain CDR2 comprising a sequence of SEQ ID NO: 6 and a heavy chain CDR3 comprising a sequence of SEQ ID NO: 7 or 8; and (b) a light chain CDR1 comprising a sequence of SEQ ID NO: 1, a light chain CDR2 comprising a sequence of SEQ ID NO: 2 and a light chain CDR3 comprising a sequence of SEQ ID NO: 4; (c) a light chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 10; and (d) a heavy chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 14, wherein the heavy chain variable domain comprises a valine at position 5, a valine at position 68, a threonine at position 91, and a lysine at on 101.

15. A method for ex vivo proliferation or isolation of activated T cells, the method comprising: contacting a population of T cells with an anti1BB antibody or antigen-binding fragment sing: (a) a heavy chain CDR1 comprising a sequence of SEQ ID NO: 5, a heavy chain CDR2 comprising a sequence of SEQ ID NO: 6 and a heavy chain CDR3 comprising a sequence of SEQ ID NO: 7 or 8; and (b) a light chain CDR1 sing a sequence of SEQ ID NO: 1, a light chain CDR2 sing a sequence of SEQ ID NO: 2 and a light chain CDR3 comprising a sequence of SEQ ID NO: 4; (c) a light chain variable domain comprising a sequence at least 98% identical to a sequence of SEQ ID NO: 10; and (d) a heavy chain variable domain comprising a sequence at least 98% cal to a sequence of SEQ ID NO: 14, n the heavy chain variable domain comprises a valine at position 5, a valine at position 68, a threonine at position 91, and a lysine at position 101.

16. A method for isolating antigen-specific activated T cells, the method comprising: (a) culturing peripheral blood mononuclear cells (PBMC) in a medium together with a peptide of an epitope of interest and IL-2; (b) inducing 4-1BB expression in the cultured cells by adding the peptide of the epitope of interest; (c) ting the cultured cells with a surface coated with an anti1BB antibody or antigen-binding fragment of any one of claims 1-14, wherein cultured cells expressing 4- 1BB adhere to the coated surface; and (d) removing unattached cells, thereby isolating antigen-specific activated T cells.

17. The method of claim 16, wherein the activated T cells are CD8+ T cells. FIG. IA llllllllllll[III/IIIIIIIII[III/IIIIIIIIIII[III/IIIIIIIII[III/IIIIIIIIIIIIII/[IIIIIllllIll/Ill]llIIllllllllll/I/I/I/I/IlllngW\\\“kw\x\\\\\\\\\wa\\\\\\\\\\\\\\\\\\\\\\\\\VNM\W Ig1f 1 55 11 16 / \ R1 (1-55 aa) l WWWW\ \ \ :1» \\ - R2 (56-110 aa) R3 (110-167 aa) R1 I 1 R2 l I -—-——---I R1.1 (1-45 aa) --—-‘ L___B§___