WO2021101346A1

WO2021101346A1 - Anti-ror1/anti-4-1bb bispecific antibodies and uses thereof

Info

Publication number: WO2021101346A1
Application number: PCT/KR2020/016567
Authority: WO
Inventors: Jongho Cho; Hyounmie Doh; Dongsop LEE; Eun Ko; Yangsoon Lee; Kyeongsu PARK; Hyejin Chung; Kyungjin PARK; Ui-Jung Jung; Jaehyoung JEON; Youngkwang Kim; Juhee Kim; Wonjun Son
Original assignee: Dong-A St Co., Ltd.; Abl Bio Inc.
Priority date: 2019-11-21
Filing date: 2020-11-23
Publication date: 2021-05-27

Abstract

The present disclosure provides an anti-ROR1/anti-4-1BB bispecific antibody capable of simultaneously binding to ROR1 and 4-1BB with high affinity, and capable of enhancing immune response and/or treating tumor (cancer) in a mammal.

Description

ANTI-ROR1/ANTI-4-1BB BISPECIFIC ANTIBODIES AND USES THEREOF

Provided is an anti-ROR1/anti-4-1BB bispecific antibody, and pharmaceutical uses thereof.

ROR (Receptor Tyrosine Kinase-Like Orphan Receptor) is a transmembrane protein of RTK (Receptor Tyrosine Kinase) family, and there are ROR1 and ROR2. ROR1 and ROR2 have 58% amino acid sequence identity and the theoretical molecular weight of two proteins is about 104kDa, but the molecular weight of ROR1 is about 103kDa, due to many N-glycosylated sites. The extracellular domain of ROR family consists of Ig, cysteine-rich, and kringle domain, and the intracellular domain consists of tyrosine kinase, Ser/Thr rich, proline rich domain. In an aspect of biological properties, the ligand of ROR2 is Wnt5a, but the ligand of ROR1 has not been discovered yet. In addition, it is presumed that ROR2 has the kinase activity, but on the other hand, ROR1 is pseudokinase. It has been known that phosphorylation of ROR1 itself is closely related to the activity of Met.

ROR1 is expressed in the process of embryo and fetal development, and controls cell polarity, cell migration and neurite growth, etc. The expression is gradually reduced according to progress of development, and it is hardly expressed in adults, and it is temporarily expressed in the process of development of B cell, and only little expression has been reported in adipocytes.

However, as the overexpression of ROR1 is observed in various cancer cells, it is classified as an oncofetal gene. In particular, ROR1 has received attention as an anti-cancer antibody target, as it is discovered that ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). It has been reported as overexpressed in not only hematologic malignancy such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), Diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL), etc. in addition to chronic lymphocytic leukemia (CLL) but also solid cancer including breast cancer, renal cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer, etc. It has been known that the expression of ROR1 in such cancers is related to poor prognosis in cancer patients and affects cancer metastasis. It has been shown that the survival time is increased and the degree of metastasis is also reduced, when a cancer cell in which the expression of ROR1 is inhibited is injected to a mouse.

Such a cancer cell-specific expression of ROR1 shows that ROR1 can be an effective cancer target, and therefore the development of an antibody specifically recognizing it is required.

Since antibodies against the same ROR1 antigen can be developed to various anti-cancer antibodies depending on properties or uses of each antibody, considering cancer-specific expression of ROR1, and its expression in various cancers, it is necessary to develop various antibodies that can replace or complement existing antibodies.

4-1BB is a member of TNF-receptor superfamily (TNFRSF) and is a costimulatory molecule which is expressed following the activation of immune cells, both innate and adaptive immune cells. 4-1BB plays important role in modulate the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, secretion of cytokines and cytolytic activity CD8 T cells. Many other studies showed that activation of 4-1BB enhances immune response to eliminate tumors in mice. Therefore, it suggests that 4-1BB is a promising target molecule in cancer immunology. Despite of their anti-tumor efficacy, anti-4-1BB antibody induced severe liver toxicity in clinical application.

The present disclosure provides an anti-ROR1/anti-4-1BB bispecific antibody capable to simultaneously bind to ROR1 and 4-1BB. The anti-ROR1/anti-4-1BB bispecific antibody may possess high affinities to ROR1 and/or 4-1BB, and be capable of enhancing immune response and/or treating tumor (cancer) in a mammal.

One embodiment provides an anti-ROR1/anti-4-1BB bispecific antibody, comprising:

(1) an anti-ROR1 antibody or an antigen-binding fragment thereof, as a ROR1 targeting moiety, which is capable of specifically recognizing and/or binding to ROR1 protein; and

(2) an anti-4-1BB antibody or an antigen-binding fragment thereof, as a 4-1BB targeting moiety, which is capable of specifically recognizing and/or binding to 4-1BB protein.

Another embodiment provides a pharmaceutical composition comprising the bispecific antibody. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be used for treating and/or preventing a cancer and/or for enhancing immune response.

Another embodiment provides a pharmaceutical composition for treating and/or preventing a cancer and/or for enhancing immune response, the composition comprising the bispecific antibody as an active ingredient.

Another embodiment provides a method of treating and/or preventing a cancer, comprising administering a pharmaceutically effective amount of the bispecific antibody or the pharmaceutical composition to a subject in need of treating and/or preventing a cancer. The method may further comprise a step of identifying the subject in need of treating and/or preventing a cancer, prior to the administering step.

Another embodiment provides a method of enhancing immune response in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the bispecific antibody or the pharmaceutical composition to the subject. The method may further comprise a step of identifying the subject in need of enhancing immune response, prior to the administering step.

Another embodiment provides a use of the bispecific antibody or the pharmaceutical composition in treating and/or preventing a cancer. Another embodiment provides a use of the bispecific antibody in preparing a medicament for treating and/or preventing a cancer.

Another embodiment provides a use of the bispecific antibody or the pharmaceutical composition in enhancing immune response. Another embodiment provides a use of the bispecific antibody in preparing a medicament for enhancing immune response.

Another embodiment provides a polynucleotide encoding the bispecific antibody.

Another embodiment provides a recombinant vector comprising the polynucleotide. The recombinant vector may be used as an expression vector of a polynucleotide encoding the bispecific antibody.

Another embodiment provides a cell comprising a polynucleotide encoding the bispecific antibody. The cell may be a recombinant cell transfected with a recombinant vector comprising the polynucleotide.

Another embodiment provides a method of preparing the bispecific antibody, comprising expressing the polynucleotide in a cell. The step of expressing the polynucleotide may be conducted by culturing the cell comprising the polynucleotide (for example, in a recombinant vector) under a condition allowing the expression of the polynucleotide.

Definitions

As used herein, the term “a” or “an” entity may refer to one or more of that entity, for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

As used herein, the term ‘consisting of a sequence,’ ‘consisting essentially of a sequence,’ or ‘comprising a sequence’ may refer to any case comprising the sequence, but it may not be intended to exclude a case comprising further sequence other than the sequence.

As used herein, the term ‘a protein or polypeptide comprising or consisting of an amino acid sequence identified by SEQ ID NO’ and ‘a gene or polynucleotide comprising or consisting of a nucleic acid sequence identified by SEQ ID NO’ may refer to a protein (or polypeptide) or gene (or polynucleotide), which consists essentially of the amino acid sequence or nucleic acid sequence, or which has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence or nucleic acid sequence with maintaining its inherent activity and/or function.

As used herein, the term “antibody” may encompass various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γl-γ4), and light chains are classified as either kappa or lambda (Κ, λ). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

The term “heavy chain” refers to a full-length heavy chain or a fragment thereof, including a variable region VH that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, CH1, CH2, and CH3, and a hinge. The term "light chain" refers to a full-length light chain or a fragment thereof, including a variable region VL that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.

The term "complementarity determining region (CDR)" refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide residues that play an important role in the binding of antibodies to an antigens or epitope. The terms "specifically binding" or "specifically recognized" is well known to one of ordinary skill in the art, and indicates that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

In this disclosure, the antibody may include, but not be limited to, polyclonal or monoclonal; and/or human, humanized, animal (e.g., mouse, rabbit, etc.) derived antibody, or chimeric antibodies (e.g., mouse-human chimeric antibody).

An animal-derived antibody which is produced by immunizing an animal with a desired antigen may generally trigger an immune rejection response when administered to humans for treatment purpose, and a chimeric antibody has been developed to suppress such immune rejection response. A chimeric antibody is formed by replacing the constant region of an animal-derived antibody, which is a cause of anti-isotype response, with the constant region of a human antibody using genetic engineering methods. The chimeric antibody has considerably improved anti-isotype response in comparison with animal-derived antibodies, but animal-derived amino acids are still present in its variable regions and thus it still contains potential side effects resulting from an anti-idiotypic response. It is a humanized antibody that has been thus developed to improve such side effects. This is manufactured by grafting CDR (complementarity determining regions) which, of the variable regions of a chimeric antibody, has an important role in antigen binding into a human antibody framework.

As used herein, the term “antigen binding fragment” refers to a fragment derived from a full immunoglobulin structure comprising a portion capable of binding to an antigen such as CDRs. For example, the antigen binding fragment may be scFv, (scFv)₂, Fab, Fab', or F(ab')₂, but not be limited thereto. In the present disclosure, the antigen binding fragment may be a fragment derived from an antibody, comprising at least one complementarity determining region, for example, selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2.

Of the antigen binding fragments, Fab is a structure having variable regions of a light chain and a heavy chain, a constant region of the light chain, and the first constant region (C_H1) of the heavy chain, and it has one antigen binding site.

Fab’ is different from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminal of heavy chain C_H1 domain. An F(ab')₂antibody is formed through disulfide bond of the cysteine residues at the hinge region of Fab’.

Fv is a minimal antibody piece having only a heavy chain variable region and light chain variable region, and a recombinant technique for producing the Fv fragment is well known in the pertinent art. Two-chain Fv may have a structure in which the heavy chain variable region is linked to the light chain variable region by a non-covalent bond, and single-chain Fv (scFv) may generally have a dimer structure as in the two-chain Fv in which the variable region of a heavy chain and the variable region of a light chain are covalently linked via a peptide linker or they are directly linked to each other at the C-terminal thereof.

The antigen binding fragments may be obtained using proteases (for example, a whole antibody is digested with papain to obtain Fab fragments, and is digested with pepsin to obtain F(ab')₂ fragments), and may be prepared by a genetic recombinant technique.

Immunoglobulin (e.g., a human immunoglobulin) or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.), class (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, IgA1, IgA2, etc.), or subclass of immunoglobulin molecule.

In the antibody or antibody fragment, portions(e.g., constant regions) except the CDRs or variable regions may be derived from a human antibody and particularly, they may be derived from IgG, IgA, IgD, IgE, IgM, or IgY, for example, IgG1, IgG2, IgG 3, or IgG4.

The antibody or antigen binding fragment may be chemically or recombinantly synthesized (not naturally occurring).

ROR1 targeting moiety

The anti-ROR1/anti-4-1BB bispecific antibody may comprise an anti-ROR1 antibody or an antigen-binding fragment thereof as a ROR1 targeting moiety.

In an embodiment, the anti- ROR1 antibody or fragment thereof can specifically bind to ROR1 (e.g., human ROR1) protein.

The ROR1 (Receptor Tyrosine Kinase-Like Orphan Receptor), that is recognized by the antibody or antigen-binding fragment thereof described herein, may refer to a transmembrane protein of an RTK (Receptor Tyrosine Kinase) family. In one embodiment, it particularly recognizes an extracellular domain. The ROR1 which the antibody recognizes may be an extracellular domain which is present in a cell membrane or is not present in a cell membrane. The human protein of ROR1 consists of 937 amino acids, and the amino acid sequence is NCBI Reference Sequence ID: NP_005003.2, and the nucleic acid sequence is NM_005012.3. Unless apparent from the context used herein, the ROR1 refers to a human hROR1, but the antibody has the binding capacity to mouse ROR1 specifically. The mouse ROR1 amino acid sequence is represented by GenBank: BAA75480.1.

The anti-ROR1 antibody or antigen-binding fragment thereof may exhibit potent binding activities to ROR1, and be useful for therapeutic and/or diagnostics uses.

In an embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 105;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 106;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, or 23;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 107;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 108; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 109.

In a specific embodiment, the amino acid sequence of SEQ ID NO: 105 may be selected from the group consisting of amino acid sequences of SEQ ID NO: 1, 2, 3, 4, and 5;

the amino acid sequence of SEQ ID NO: 106 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12, 13, and 14;

the amino acid sequence of SEQ ID NO: 107 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 24, 25, 26, 27, 28, 29, 30, and 31;

the amino acid sequence of SEQ ID NO: 108 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 32, 33, 34, 35, 36, 37, 38, and 39; and

the amino acid sequence of SEQ ID NO: 109 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 40, 41, 42, 43, and 44.

For example, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 31;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, or 39; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 40, 41, 42, 43, or 44.

The amino acid sequences of the CDRs of the anti-ROR1 antibody or an antigen-binding fragment are listed in Table 1.

Examples of CDRs of anti-ROR1 antibody or antigen-binding fragment thereof according to Kabat definition

Description	Amino acid Sequence	SEQ ID NO
CDR-H	SYDMS	1
	DYYMS	2
	NYDMS	3
	NYAMS	4
	DYDMS	5
General Formula of CDR-H1	X1YX2MS, wherein X1 is S, D, or N; and X2 is D, Y, or A	105
CDR-H2	WISPDSGSIYYADSVKG	6
	SISPDGSNTYYADSVKG	7
	WISPGGGSKYYADSVKG	8
	AIYHSGSSKYYADSVKG	9
	GISHGSGNKYYADSVKG	10
	SISHNSGSTYYADSVKG	11
	VISPDGGSIYYADSVKG	12
	SISPSSGSSIYYADSVKG	13
	SISPDASNTYYADSVKG	14
General Formula of CDR-H2	X3IX4X5X6X7X8X9X10YYADSVKG, wherein X3 is W, S, A, G, or V; X4 is S or Y; X5 is P or H; X6 is D, G, S, or N; X7 is S, G, or A; X8 is G or S; X9 is S or N; and X10 is I, T, or K	106
CDR-H3	PTGRFDY	15
	NLRAFDY	16
	VNGRFDY	17
	GGNGAWDTGFDY	18
	RLSLRRRPSYYSDNAMDV	19
	FISARKSLGRSYSNGMDV	20
	DVVECNMNPCSYDNAMDV	21
	APGWCQAPSCYYDNAMDV	22
	GGNAAWDTGFDY	23
CDR-L1	SGSSSNIGNNNVN	24
	SGSSSNIGSNTVY	25
	SGSSSNIGNNNVS	26
	SGSSSNIGSNDVS	27
	TGSSSNIGNNAVN	28
	TGSSSNIGSNDVT	29
	SGSSSNIGSNYVS	30
	SGSSSNIGNNDVS	31
General Formula of CDR-L1	X11GSSSNIGX12NX13VX14, wherein X11 is S or T; X12 is N or S; X13 is N, T, D, A, or Y; and X14 is N, Y, S, or T	107
CDR-L2	YDNKRPS	32
	ANSQRPS	33
	ADSHRPS	34
	YDNNRPS	35
	YDSNRPS	36
	ADSKRPS	37
	DDSHRPS	38
	DDSQRPS	39
General Formula of CDR-L2	X15X16X17X18RPS, wherein X15 is Y, A, or D; X16 is D or N; X17 is N or S; and X18 is K, Q, H, or N;	108
CDR-L3	GTWDASLSGYV	40
CDR-L3	GSWDYSLSGYV	41
	ATWDYSLSGYV	42
	GAWDDSLSGYV	43
	GTWDYSLSGYV	44
General Formula of CDR-L3	X19X20WDX21SLSGYV, wherein X19 is G or A; X20 is T, S, or A; and X21 is A, Y, or D	109

In one embodiment, the anti-ROR1 antibody or an antigen binding fragment thereof can be designed by suitably mixing and matching the CDRs listed in Table 1 so as to maintain its affinity to ROR1.For example, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

(1) a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 6, a CDR-H3 of SEQ ID NO: 15, a CDR-L1 of SEQ ID NO: 24, a CDR-L2 of SEQ ID NO: 32, and a CDR-L3 of SEQ ID NO: 40;

(2) a CDR-H1 of SEQ ID NO: 2, a CDR-H2 of SEQ ID NO: 7, a CDR-H3 of SEQ ID NO: 16, a CDR-L1 of SEQ ID NO: 25, a CDR-L2 of SEQ ID NO: 33, and a CDR-L3 of SEQ ID NO: 41;

(3) a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 8, a CDR-H3 of SEQ ID NO: 17, a CDR-L1 of SEQ ID NO: 26, a CDR-L2 of SEQ ID NO: 34, and a CDR-L3 of SEQ ID NO: 42;

(4) a CDR-H1 of SEQ ID NO: 3, a CDR-H2 of SEQ ID NO: 9, a CDR-H3 of SEQ ID NO: 18, a CDR-L1 of SEQ ID NO: 27, a CDR-L2 of SEQ ID NO: 35, and a CDR-L3 of SEQ ID NO: 43;

(5) a CDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 10, a CDR-H3 of SEQ ID NO: 19, a CDR-L1 of SEQ ID NO: 28, a CDR-L2 of SEQ ID NO: 36, and a CDR-L3 of SEQ ID NO: 43;

(6) a CDR-H1 of SEQ ID NO: 4, a CDR-H2 of SEQ ID NO: 11, a CDR-H3 of SEQ ID NO: 20, a CDR-L1 of SEQ ID NO: 29, a CDR-L2 of SEQ ID NO: 37, and a CDR-L3 of SEQ ID NO: 44;

(7) a CDR-H1 of SEQ ID NO: 5, a CDR-H2 of SEQ ID NO: 12, a CDR-H3 of SEQ ID NO: 21, a CDR-L1 of SEQ ID NO: 30, a CDR-L2 of SEQ ID NO: 38, and a CDR-L3 of SEQ ID NO: 43;

(8) a CDR-H1 of SEQ ID NO: 3, a CDR-H2 of SEQ ID NO: 13, a CDR-H3 of SEQ ID NO: 22, a CDR-L1 of SEQ ID NO: 31, a CDR-L2 of SEQ ID NO: 39, and a CDR-L3 of SEQ ID NO: 43;

(9) a CDR-H1 of SEQ ID NO: 2, a CDR-H2 of SEQ ID NO: 14, a CDR-H3 of SEQ ID NO: 16, a CDR-L1 of SEQ ID NO: 25, a CDR-L2 of SEQ ID NO: 33, and a CDR-L3 of SEQ ID NO: 41; or

(10) a CDR-H1 of SEQ ID NO: 3, a CDR-H2 of SEQ ID NO: 9, a CDR-H3 of SEQ ID NO: 23, a CDR-L1 of SEQ ID NO: 27, a CDR-L2 of SEQ ID NO: 35, and a CDR-L3 of SEQ ID NO: 43.

a heavy chain variable region comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 105, a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 106, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, or 23; and

a light chain variable region comprising a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 107, a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 108, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 109.

More specifically, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

a heavy chain variable region comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5, a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, or 23; and

a light chain variable region comprising a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 31, a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, or 39, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 40, 41, 42, 43, or 44.

Non-limiting examples of the anti-ROR1 antibody or antigen-binding fragment may comprise:

a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences; and

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, or 63, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences.

Examples of heavy chain variable regions and light chain variable regions of the antibody or antigen-binding fragment, which comprise the light chain CDR and heavy chain CDR sequences in Table 1, are listed in the following Table 2.

Examples of variable regions of anti-ROR1 antibody or antigen-binding fragment thereof

Heavy Chain Variable Region (VH) Sequence	SEQ ID NO
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISPDSGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPTGRFDYWGQGTLVTVSS	45
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSS	46
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISPGGGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNGRFDYWGQGTLVTVSS	47
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSS	48
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSGISHGSGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRLSLRRRPSYYSDNAMDVWGQGTLVTVSS	49
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISHNSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFISARKSLGRSYSNGMDVWGQGTLVTVSS	50
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYDMSWVRQAPGKGLEWVSVISPDGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVVECNMNPCSYDNAMDVWGQGTLVTVSS	51
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSSISPSSGSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGWCQAPSCYYDNAMDVWGQGTLVTVSS	52
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDASNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSS	53
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNAAWDTGFDYWGQGTLVTVSS	54
Light Chain Variable Region (VL) Sequence	SEQ ID NO
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNNVNWYQQLPGTAPKLLIYYDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDASLSGYVFGGGTKLTVLG	55
QSVLTQPPPASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG	56
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNNVSWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVLG	57
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	58
QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNAVNWYQQLPGTAPKLLIYYDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	59
QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNDVTWYQQLPGTAPKLLIYADSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDYSLSGYVFGGGTKLTVLG	60
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLIYDDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	61
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVSWYQQLPGTAPKLLIYDDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	62
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG	63

In other embodiment, the variable regions of heavy chain and light chain disclosed in Table 2 can be suitably combined (mixed and marched) for preparation of various forms of antibodies, and for example, they can form a single-chain antibody such as ScFV, or domain antibody, or full-length antibody (e.g., an IgG form antibody comprising two heavy chains and two light chains).For example, the anti-ROR1 antibody or antigen-binding fragment may comprise:

(1) a heavy chain variable region of SEQ ID NO: 45 and a light chain variable region of SEQ ID NO: 55;

(2) a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 56;

(3) a heavy chain variable region of SEQ ID NO: 47 and a light chain variable region of SEQ ID NO: 57;

(4) a heavy chain variable region of SEQ ID NO: 48 and a light chain variable region of SEQ ID NO: 58;

(5) a heavy chain variable region of SEQ ID NO: 49 and a light chain variable region of SEQ ID NO: 59;

(6) a heavy chain variable region of SEQ ID NO: 50 and a light chain variable region of SEQ ID NO: 60;

(7) a heavy chain variable region of SEQ ID NO: 51 and a light chain variable region of SEQ ID NO: 61;

(8) a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 62;

(9) a heavy chain variable region of SEQ ID NO: 53 and a light chain variable region of SEQ ID NO: 63; or

(10) a heavy chain variable region of SEQ ID NO: 54 and a light chain variable region of SEQ ID NO: 58.

Each of heavy chain variable regions and light chain variable regions disclosed herein may be combined with various constant regions of heavy chain and light chain to form heavy chain and light chain of an intact antibody, respectively.

a heavy chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 64, or 65, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences; and

a light chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 66 or 67, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences.

In another embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof may be a scFv (single chain variable fragment) comprising:

wherein the heavy chain variable region and the light chain variable region may be linked to each other in any order directly (i.e., without a linker) or via a peptide linker.

More specifically, the anti-ROR1 antibody or antigen-binding fragment thereof may be a scFv comprising:

a light chain variable region comprising a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 31, a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, or 39, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 40, 41, 42, 43, or 44,

wherein the heavy chain variable region and the light chain variable region may be linked to each other in any order directly or via a peptide linker.

For example, the anti-ROR1 antibody or antigen-binding fragment thereof may be a scFv comprising:

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, or 63, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences,

(10) a heavy chain variable region of SEQ ID NO: 54 and a light chain variable region of SEQ ID NO: 58,

In the present disclosure, the anti-ROR1 scFv may comprise a heavy chain variable region and a light chain variable region, in any order. For example, the anti-ROR1 scFv may comprise a light chain variable region and a heavy chain variable region in order from N-terminus to C-terminus. Alternatively, the anti-ROR1 scFv may comprise a heavy chain variable region and a light chain variable region, in order from N-terminus to C-terminus.

In an embodiment, in the scFv, the heavy chain variable region and the light chain variable region may be linked via a suitable peptide linker. For example, the peptide linker may be (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9.

For example, binding affinities of an anti-ROR1 antibody or an antigen binding fragment thereof of the disclosure to human ROR1 include those with a dissociation constant or KD of 1x10^-6 M or less, 1x10^-7 M or less, 1x10^-8 M or less, 1x10^-9 M or less, or 1x10^-10 M or less.

4-1BB targeting moiety

The anti-ROR1/anti-4-1BB bispecific antibody may comprise an anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety.

In an embodiment, the anti-4-1BB antibody or fragment thereof can specifically bind to 4-1BB (e.g., human 4-1BB) protein.

For example, the human 4-1BB protein may be selected from the group consisting of proteins represented by NCBI Accession No. NP_001552.2, etc., but may not be limited thereto. These anti-4-1BB antibodies or antigen-binding fragments thereof are capable of enhancing immune response and/or treating tumor (cancer) in a mammal. The anti-4-1BB antibody or an antigen-binding fragment thereof is characterized by being localized and/or activated in tumor microenvironment (TME) and/or considerably reducing liver toxicities compared to pre-existing anti-4-1BB antibodies, with maintaining the efficacies of enhancing immune response enhancement and/or tumor treatment.

The term “4-1BB” refers to CD137, or TNFRSF9 (TNF Receptor 25 Superfamily Member 9), is a member of TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule which is expressed following the activation of immune cells, both innate and adaptive immune cells. As used herein, 4-1BB may be originated from a mammal, for example, Homo sapiens (human) (NCBI Accession No. NP_001552.2).

As described herein, the term “4-1BB” includes variants, isoforms, homologs, orthologs, and paralogs. For example, antibodies specific for a human 4-1BB protein may, in certain cases, cross-react with a 4-1BB protein from a species other than human. In other embodiments, the antibodies specific for a human 4-1BB protein may be completely specific for the human 4-1BB protein and may exhibit species or other types of cross-reactivity, or may cross-react with 4-1BB from certain other species but not all other species (e.g., cross-react with monkey 4-1BB, but not mouse 4-1BB). The term “human 4-1BB” refers to human sequence 4-1BB, such as the complete amino acid sequence of human 4-1BB having NCBI Accession No. NP_001552.2. The term “mouse 4-1BB” refers to mouse sequence 4-1BB, such as the complete amino acid sequence of mouse 4-1BB having NCBI Accession No. NP 033430.1. 4-1BB also can be known in the art as, for example, CD137. The human 4-1BB sequence in the disclosure may differ from human 4-1BB of NCBI Accession No. NP_001552.2 by having, e.g., conserved mutations or mutations in non-conserved regions and the 4-1BB in the disclosure has substantially the same biological function as the human 4-1BB of NCBI Accession No. NP_001552.2.

In an embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof may comprise:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 110;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 111;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 83 or 73;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 76;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 77; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 78.

In a specific embodiment, the amino acid sequence of SEQ ID NO: 110 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 68 and 69;

the amino acid sequence of SEQ ID NO: 111 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 70 and 71; and

the amino acid sequence of SEQ ID NO: 83 may be selected from the group consisting of amino acid sequences of SEQ ID NOS: 72, 74 and 75.

For example, the anti-4-1BB antibody or antigen-binding fragment thereof may comprise:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 68 or 69;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 70 or 71;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 72, 73, 74, or 75;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 76;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 77; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 78.

The amino acid sequences of the CDRs of the anti-ROR1 antibody or an antigen-binding fragment are listed in Table 3.

Examples of CDRs of anti-4-1BB antibody or antigen-binding fragment thereof according to Kabat definition

Description	Amino Acid Sequence	SEQ ID NO:
CDR-H1	SYDMS	68
CDR-H1	GYDMS	69
General Formula of CDR-H1	X22YDMS, wherein X22 is S or G	110
CDR-H2	WISYSGGSIYYADSVKG		70
CDR-H2	VIYPDDGNTYYADSVKG	71
General Formula of CDR-H2	X23IX24X25X26X27GX28X29YYADSVKG, wherein X23 is W or V; X24 is S or Y; X25 is Y or P; X26 is S or D; X27 is G or D; X28 is S or N; and X29 is I or T	111
CDR-H3	DGQRNSMREFDY	72
	HGGQKPTTKSSSAYGMDG	73
	DAQRNSMREFDY	74
	DAQRQSMREFDY	75
General Formula of CDR-H3 (except for 1A12 and 1A12M1)	DX29QRX30SMREFDY, wherein X29 is G or A; and X30 is N or Q	83
CDR-L1	SGSSSNIGNNYVT	76
CDR-L2	ADSHRPS	77
CDR-L3	ATWDYSLSGYV	78

In one embodiment, the anti-4-1BB antibody or an antigen binding fragment thereof can be designed by suitably mixing and matching the CDRs listed in Table 3 so as to maintain its affinity to 4-1BB.For example, the anti-4-1BB antibody or an antigen binding fragment thereof may comprise:

(1) a CDR-H1 of SEQ ID NO: 68, a CDR-H2 of SEQ ID NO: 70, a CDR-H3 of SEQ ID NO: 72, a CDR-L1 of SEQ ID NO: 76, a CDR-L2 of SEQ ID NO: 77, and a CDR-L3 of SEQ ID NO: 78;

(2) a CDR-H1 of SEQ ID NO: 68, a CDR-H2 of SEQ ID NO: 70, a CDR-H3 of SEQ ID NO: 74, a CDR-L1 of SEQ ID NO: 76, a CDR-L2 of SEQ ID NO: 77, and a CDR-L3 of SEQ ID NO: 78;

(3) a CDR-H1 of SEQ ID NO: 68, a CDR-H2 of SEQ ID NO: 70, a CDR-H3 of SEQ ID NO: 75, a CDR-L1 of SEQ ID NO: 76, a CDR-L2 of SEQ ID NO: 77, and a CDR-L3 of SEQ ID NO: 78; or

(4) a CDR-H1 of SEQ ID NO: 69, a CDR-H2 of SEQ ID NO: 71, a CDR-H3 of SEQ ID NO: 73, a CDR-L1 of SEQ ID NO: 76, a CDR-L2 of SEQ ID NO: 77, and a CDR-L3 of SEQ ID NO: 78.

a heavy chain variable region comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 110, a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 111, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 83 or 73; and

a light chain variable region comprising a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 76, a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 77, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 78.

a heavy chain variable region comprising a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 68 or 69, a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 70 or 71, and a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 72, 73, 74, or 75; and

Non-limiting examples of the anti-4-1BB antibody or fragment thereof may comprise:

a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 79, 80, 81, 82, 84, 98, 99. 100, 101, or 102, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences; and

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 85, 86, 103, or 104, or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences.

Examples of heavy chain variable regions and light chain variable regions of the antibody or antigen-binding fragment, which comprise the light chain CDR and heavy chain CDR sequences in Table 3, are listed in the following Table 4.

Examples of variable regions of anti-4-1BB antibody or antigen binding fragment thereof

Heavy Chain Variable Region (VH) Sequence	SEQ ID NO
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGQRNSMREFDYWGQGTLVTVSS	79
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	80
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS	82
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	84
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGQRNSMREFDYWGQGTLVTVSS	98
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	99
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS	100
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKGLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	101
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKGLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	102
Light Chain Variable Region (VL) Sequence	SEQ ID NO
QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	85
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	86
QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	103
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	104

In other embodiment, the variable regions of heavy chain and light chain disclosed in Table 4 can be suitably combined (mixed and marched) for preparation of various forms of antibodies, and for example, they can form a single-chain antibody such as ScFV, or domain antibody, or full-length antibody (e.g., an IgG form antibody comprising two heavy chains and two light chains).

For example, the anti-4-1BB antibody or antigen-binding fragment may comprise:

(1) a heavy chain variable region of SEQ ID NO: 79 and a light chain variable region of SEQ ID NO: 85;

(2) a heavy chain variable region of SEQ ID NO: 80 and a light chain variable region of SEQ ID NO: 85;

(3) a heavy chain variable region of SEQ ID NO: 81 and a light chain variable region of SEQ ID NO: 85;

(4) a heavy chain variable region of SEQ ID NO: 81 and a light chain variable region of SEQ ID NO: 86;

(5) a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 85;

(6) a heavy chain variable region of SEQ ID NO: 82 and a light chain variable region of SEQ ID NO: 86;

(7) a heavy chain variable region of SEQ ID NO: 84 and a light chain variable region of SEQ ID NO: 86;

(8) a heavy chain variable region of SEQ ID NO: 98 and a light chain variable region of SEQ ID NO: 103;

(9) a heavy chain variable region of SEQ ID NO: 99 and a light chain variable region of SEQ ID NO: 103;

(10) a heavy chain variable region of SEQ ID NO: 100 and a light chain variable region of SEQ ID NO: 103;

(11) a heavy chain variable region of SEQ ID NO: 99 and a light chain variable region of SEQ ID NO: 104;

(12) a heavy chain variable region of SEQ ID NO: 100 and a light chain variable region of SEQ ID NO: 104;

(13) a heavy chain variable region of SEQ ID NO: 101 and a light chain variable region of SEQ ID NO: 103; or

(14) a heavy chain variable region of SEQ ID NO: 101 and a light chain variable region of SEQ ID NO: 104.

In another embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof may be a scFv (single chain variable fragment) comprising:

a light chain variable region comprising a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 76, a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 77, and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 78,

More specifically, the anti-4-1BB antibody or antigen-binding fragment thereof may be a scFv comprising:

For example, the anti-4-1BB antibody or fragment thereof may be a scFv comprising:

a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 79, 80, 81, 82, 84, 98, 99. 100, 101, or 102 (e.g., SEQ ID NO: 79, 80, 81, 82, or 84), or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences; and

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 85, 86, 103, or 104 (e.g., SEQ ID NO: 85 or 86), or an amino acid sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the above described amino acid sequences,

For example, the anti-4-1BB antibody or antigen-binding fragment thereof may be a scFv comprising:

(14) a heavy chain variable region of SEQ ID NO: 101 and a light chain variable region of SEQ ID NO: 104,

In the present disclosure, the anti-4-1BB scFv may comprise a heavy chain variable region and a light chain variable region, in any order. For example, the anti-4-1BB scFv may comprise a light chain variable region and a heavy chain variable region in order from N-terminus to C-terminus. Alternatively, the anti-4-1BB scFv may comprise a heavy chain variable region and a light chain variable region in order from N-terminus to C-terminus.

These anti-4-1BB antibodies may be useful for therapeutic purposes such as treating various types of cancer, etc., and can also be used for diagnostic and prognostic purposes.

The antibodies of the disclosure are characterized by particular functional features or properties of the antibodies. For example, the antibodies specifically bind to human 4-1BB and may bind to 4-1BB originated from certain other species, e.g., monkey 4-1BB, e.g., cynomolgus monkey, rhesus monkey, but may not substantially bind to 4-1BB originated from certain other species, e.g., mouse 4-1BB. Preferably, an antibody of the disclosure binds to human 4-1BB with high affinity.

The binding of an antibody of the disclosure to 4-1BB can be assessed using one or more techniques well established in the art. For example, in a preferred embodiment, an antibody can be tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human 4-1BB, such as CHO cells that have been transfected to express 4-1BB, e.g., human 4-1BB, or monkey 4-1BB, e.g., rhesus or cynomolgus monkey or mouse 4-1BB on their cell surface. Other suitable cells for use in flow cytometry assays include anti-CD3-stimulated CD4⁺ activated T cells, which express native 4-1BB. Still other suitable binding assays include ELISA assays, for example using a recombinant 4-1BB protein. Additionally, or alternatively, the binding of the antibody, including the binding kinetics (e.g., KD value) can be tested in Octet analysis. For example, binding affinities of an anti-4-1BB antibody or an antigen binding fragment thereof of the disclosure to human 4-1BB include those with a dissociation constant or KD of 1x10^-6 M or less, 1x10^-7 M or less, 1x10^-8 M or less, 1x10^-9 M or less, 1x10^-10 M or less, or 1.80 x 10^-10 M or less.

Constant Region of Antibodies

In some embodiments, the anti-4-1BB antibody or fragment thereof, the anti-ROR1 antibody or fragment thereof, and/or the bispecific antibody may comprise a heavy chain constant region, a light chain constant region, an Fc region, or the combination thereof, in addition to a heavy chain variable region and a light chain variable region as described above.

In some embodiments, the light chain constant region may be a kappa or lambda constant region.

In some embodiments, the antibody is of an isotype of IgG, IgM, IgA, IgE or IgD, for example, human IgG, human IgM, human IgA, human IgE, or human IgD. In some embodiments, the isotype may be IgG, for example human IgG, such as, IgG1, IgG2, IgG3, or IgG4. In some embodiments, the fragment (antigen-binding fragment of the anti-ROR1 antibody) may be any fragment comprising heavy chain CDRs and/or light chain CDRs of the antibody, and for example, it may be selected from the group consisting of Fab, Fab', F(ab')₂, Fd (comprising a heavy chain variable region and a CH1 domain), Fv (a heavy chain variable region and/or a light chain variable region), single-chain Fv (scFv; comprising or consisting essentially of a heavy chain variable region and a light chain variable region, in any order, and a peptide linker between the heavy chain variable region and the light chain variable region), single-chain antibodies, disulfide-linked Fvs (sdFv), and the like.

For example, the constant regions may comprise the following amino acid sequences listed in Table 5:

	Amino acid sequence (N→C)	SEQ ID NO
Heavy Chain Constant Region (WT IgG1)	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	89
Heavy Chain Constant Region (NA, IgG1 with N297A mutation)	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	90
Light Chain Constant Region	QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS	91

Without limitation, the anti-4-1BB antibody or fragment thereof, the anti-ROR1 antibody or fragment thereof, and/or the bispecific antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, antibody or fragment thereof is not naturally occurring, or chemically or recombinantly synthesized.Given that each of these antibodies can bind to 4-1BB (such as, human 4-1BB) and/or to ROR1 (such as, human ROR1), the CDR sequences or the V_H and V_L sequences as described above can be “mixed and matched” to create other anti-4-1BB binding molecules and/or other anti-ROR1 binding molecules. Preferably, when the CDRs sequences or V_H and V_L chains are mixed and matched, for example, a V_H sequence from a particular V_H/V_L pairing is replaced with a structurally similar V_H sequence. Likewise, preferably a V_L sequence from a particular V_H/V_L pairing is replaced with a structurally similar V_L sequence.

Anti-ROR1/anti-4-1BB bispecific antibody

The bispecific antibody described herein may comprise:

(1) an anti-ROR1 antibody or an antigen-binding fragment thereof, which is capable of specifically recognizing and/or binding to ROR1 protein; and

(2) an anti-4-1BB antibody or an antigen-binding fragment thereof, which is capable of specifically recognizing and/or binding to 4-1BB protein.

The bispecific antibody can bind both of ROR1 and 4-1BB.

In the bispecific antibody, the anti-ROR1 antibody or antigen-binding fragment thereof and the anti-4-1BB antibody or antigen-binding fragment thereof are as described above.

The bispecific antibody may possess advantages due to functions of an anti-ROR1 antibody or its fragment and/or an anti-4-1BB antibody or its fragment. In certain embodiments, due to an anti-ROR1 antibody or antigen-binding fragment thereof, the bispecific antibody may possess ability to enhance immune cell proliferation, survival, secretion of cytokines and/or cytolytic activity CD8 T cells. In certain embodiments, due to an anti-4-1BB antibody or antigen-binding fragment thereof, the bispecific antibody may be capable of binding to human 4-1BB and exhibiting an ability to activate T cells. The anti-ROR1/anti-4-1BB bispecific antibody may activate 4-1BB signaling under the condition of ROR1-expressing tumor cells. In addition, the anti-4-1BB antibody or an antigen-binding fragment thereof contained in the bispecific antibody may be characterized by localizing and/or activating only in tumor microenvironment (TME), and/or considerably reducing liver toxicities compared to pre-existing anti-4-1BB antibodies, with maintaining the efficacies of immune response enhancement and/or tumor treatment.

Other means by which to evaluate the ability of the antibody to stimulate an immune response include the ability of the antibody to inhibit tumor growth, such as in an in vivo tumor graft model.

In the bispecific antibody comprising the ROR1 targeting moiety and the 4-1BB targeting moiety, one of the ROR1 targeting moiety and the 4-1BB targeting moiety can be a full-length antibody, and the other can be an antigen-binding fragment (e.g., scFv) comprising heavy chain CDRs, light chain CDRs, or a combination thereof. The full-length antibody targeting one of ROR1 and 4-1BB proteins, and the antigen-binding fragment targeting the other protein may be chemically linked (e.g., covalently linked) directly or via a peptide linker. The antigen-binding fragment (e.g., scFv) may be linked directly or via a peptide linker to N-terminus of the full-length antibody (e.g., N-terminus of a light chain or a heavy chain of the full-length antibody), C-terminus of the full-length antibody (e.g., C-terminus of a heavy chain (or Fc or CH3 domain) of the full-length antibody), or both thereof.

In an embodiment, the bispecific antibody may comprise a full-length anti-ROR1 antibody, an antigen-binding fragment (e.g., scFv) of an anti-4-1BB antibody, and a peptide linker therebetween. In other embodiment, the bispecific antibody may comprise a full-length anti-4-1BB antibody, an antigen-binding fragment (e.g., scFv) of an anti-ROR1 antibody, and a peptide linker therebetween.

In an embodiment, the scFv contained in the bispecific antibody may comprise a heavy chain variable region and a light chain variable region in any order. For example, the scFv contained in the bispecific antibody may comprise a heavy chain variable region and a light chain variable, in a direction from N-terminus to C-terminus, and optionally a peptide linker therebetween, or alternatively, the scFv contained in the bispecific antibody may comprise a light chain variable region and a heavy chain variable, in a direction from N-terminus to C-terminus, and optionally a peptide linker therebetween.

In an embodiment, the anti-ROR1/anti-4-1BB bispecific antibody activates 4-1BB signaling, and as a result immune response, depending on ROR1 expressed on cell surfaces.

When the bispecific antibody comprises a full-length anti-ROR1 antibody and an anti-4-1BB scFv, the bispecific antibody may comprise:

(i) a first polypeptide comprising, in a direction from N-terminus to C-terminus:

a heavy chain of an anti-ROR1 antibody,

optionally, a peptide linker (a first peptide linker), and

an anti-4-1BB scFv; and

(ii) a second polypeptide comprising a light chain of the anti-ROR1 antibody,

wherein the anti-4-1BB scFv may comprise, in a direction from N-terminus to C-terminus:

a light chain variable region of an anti-4-1BB antibody,

optionally, a peptide linker (a second peptide linker), and

a heavy chain variable region of the anti-4-1BB antibody.

Alternatively, the bispecific antibody may comprise:

a heavy chain of an anti-ROR1 antibody,

optionally, a peptide linker (a first peptide linker), and

an anti-4-1BB scFv; and

(ii) a second polypeptide comprising a light chain of the anti-ROR1 antibody,

a heavy chain variable region of the anti-4-1BB antibody,

optionally, a peptide linker (a second peptide linker), and

a light chain variable region of an anti-4-1BB antibody.

Alternatively, the bispecific antibody may comprise:

an anti-4-1BB scFv,

optionally, a peptide linker (a first peptide linker), and

a heavy chain of an anti-ROR1 antibody; and

(ii) a second polypeptide comprising a light chain of the anti-ROR1 antibody,

a light chain variable region of an anti-4-1BB antibody,

optionally, a peptide linker (a second peptide linker), and

a heavy chain variable region of the anti-4-1BB antibody.

Alternatively, the bispecific antibody may comprise:

an anti-4-1BB scFv,

optionally, a peptide linker (a first peptide linker), and

a heavy chain of an anti-ROR1 antibody; and

(ii) a second polypeptide comprising a light chain of the anti-ROR1 antibody,

a heavy chain variable region of the anti-4-1BB antibody,

optionally, a peptide linker (a second peptide linker), and

a light chain variable region of an anti-4-1BB antibody.

When the bispecific antibody comprises a full-length anti-4-1BB antibody and an anti-ROR1 scFv, the bispecific antibody may comprise:

a heavy chain of an anti-4-1BB antibody,

optionally, a peptide linker (a first peptide linker), and

an anti-ROR1 scFv; and

(ii) a second polypeptide comprising a light chain of the anti-4-1BB antibody,

wherein the anti-ROR1 scFv may comprise, in a direction from N-terminus to C-terminus:

a light chain variable region of an anti-ROR1 antibody,

optionally, a peptide linker (a second peptide linker), and

a heavy chain variable region of the anti-ROR1 antibody.

Alternatively, the bispecific antibody may comprise:

a heavy chain of an a anti-4-1BB antibody,

optionally, a peptide linker (a first peptide linker), and

an anti-ROR1 scFv; and

(ii) a second polypeptide comprising a light chain of the anti-4-1BB antibody,

a heavy chain variable region of the anti-ROR1 antibody,

optionally, a peptide linker (a second peptide linker), and

a light chain variable region of an anti-ROR1 antibody.

Alternatively, the bispecific antibody may comprise:

an anti-ROR1 scFv,

optionally, a peptide linker (a first peptide linker), and

a heavy chain of an anti-4-1BB antibody; and

(ii) a second polypeptide comprising a light chain of the anti-4-1BB antibody,

a light chain variable region of an anti-ROR1 antibody,

optionally, a peptide linker (a second peptide linker), and

a heavy chain variable region of the anti-ROR1 antibody.

Alternatively, the bispecific antibody may comprise:

an anti-ROR1 scFv,

optionally, a peptide linker (a first peptide linker), and

a heavy chain of an anti-4-1BB antibody; and

(ii) a second polypeptide comprising a light chain of the anti-4-1BB antibody,

a heavy chain variable region of the anti-ROR1 antibody,

optionally, a peptide linker (a second peptide linker), and

a light chain variable region of an anti-ROR1 antibody.

The first peptide linker and the second peptide linker may be, independently, present or absent in the bispecific antibody, and the same with or different from each other.

In another embodiment, both of the ROR1 targeting moiety and the 4-1BB targeting moiety contained in the bispecific antibody may be a full-length antibody or an antigen-binding fragment comprising heavy chain CDRs, light chain CDRs, or a combination thereof, which are linked to each other directly or via a peptide linker.

Given that each of antibodies can bind to both of 4-1BB (such as, human 4-1BB) and ROR1 (such as, human ROR1), the CDR sequences, or VH (heavy chain variable region) and VL (light chain variable region) sequences as disclosed herein can be “mixed and matched” to create other anti-ROR1/anti-4-1BB binding bispecific molecules.

In another embodiment, both of the ROR1 targeting moiety and the 4-1BB targeting moiety may be a full-length antibody or an antigen-binding fragment comprising heavy chain CDRs, light chain CDRs, or a combination thereof.

In another embodiment, the bispecific antibody may be in a heterodimeric form, which comprises a first arm comprising a pair of a first heavy chain and a first light chain targeting one of ROR1 and 4-1BB, and a second arm comprising a pair of a second heavy chain and a second light chain targeting the other one.

In an embodiment, the full-length antibody may be in a full-length immunoglobulin form (e.g., IgG, IgM, IgA, IgE or IgD, such as, human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')₂, Fd, Fv, scFv, single-chain antibodies, sdFv, and the like, as described above. For example, the full-length antibody may be in a full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4) form, and the antigen-binding fragment may be scFv.

Peptide linker

The use of a peptide linker for the bispecific antibody may lead to a high purity of the antibody. That is, for high purity of the antibody, the bispecific antibody may comprise a peptide linker between a heavy chain and scFv in a first polypeptide (a first peptide linker), and/or between heavy and light variable regions in scFv (a second peptide linker).

As used herein, the term “peptide linker” may be those including any amino acids of 1 to 100, particularly 2 to 50, and any kinds of amino acids may be included without any restrictions. The peptide linker may include for example, Gly, Asn and/or Ser residues, and also include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for the peptide linker may be those known in the relevant art. Meanwhile, a length of the peptide linker may be variously determined within such a limit that the functions of the polypeptide and/or scFv will not be affected. For instance, the peptide linker may be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) of one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented as (G_mS_l)_n (m, l, and n, are independently an integer of about 1 to about 10, particularly, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In one embodiment, the peptide linker can be amino acids of (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9, but not be limited thereto.

Variable antibodies

For example, an antibody described herein may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

Antibodies or variants described herein may comprise derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the antigen (e.g., an epitope). For example, but not by way of limitation, the antibodies can be modified, e.g., by at least one selected from the group consisting of glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, and the like. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

The antibodies or fragments thereof can be detectably labeled by tagging (coupling) with a conventional labeling material selected from chemiluminescent compounds, fluorescent compounds (e.g., fluorescence emitting metals), radioisotopes, dyes, etc. The presence of the tagged antibodies or fragments thereof can be detected by measuring a signal arising during a chemical reaction between the antibody (or fragment thereof) and the labeling material. Examples of particularly useful labeling material may be at least one selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, fluorescence emitting metals, and the like. For example, the fluorescence emitting metals may be ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In certain embodiments, the prepared bispecific antibodies will not elicit a deleterious immune response in the animal to be treated, e.g., in a human. In one embodiment, the bispecific antibody may be modified to reduce their immunogenicity using any conventional techniques. For example, the bispecific antibody may be a humanized, primatized, deimmunized, or chimeric antibody. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, such as, (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues.

De-immunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term “de-immunization” may include alteration of an antibody to modify T-cell epitopes (see, e.g., International Application Publication Nos. : WO/9852976 A1 and WO/0034317 A2). For example, variable heavy chain and variable light chain sequences from the starting antibody are analyzed and a human T-cell epitope “map” from each V (variable) region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence is created. Individual T-cell epitopes from the T-cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative variable heavy and variable light sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides. Typically, between 12 and 24 variant antibodies are generated and tested for binding and/or function. Complete heavy and light chain genes comprising modified variable and human constant regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

The binding specificity and/or affinity of the bispecific antibody to each target protein can be determined by any conventional assay, for example, in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunoabsorbent assay (ELISA), but not be limited thereto.

Alternatively, techniques described for the production of single-chain units can be adapted to produce single-chain units of the present disclosure. Single-chain units are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (peptide linker), resulting in a single-chain fusion peptide (scFv). Techniques for the assembly of functional Fv fragments in E. coli may also be used.

Examples of techniques which can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Pat. Nos. 4,946,778, 5,258,498, etc.). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.

Humanized antibodies are antibody molecules derived from a non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen-binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison to identify unusual framework residues at particular positions. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting, veneering or resurfacing, and chain shuffling.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies.

Completely human antibodies which recognize a selected epitope can also be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.

In another embodiment, DNA encoding desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.

Additionally, using routine recombinant DNA techniques, one or more of the CDRs of the bisprcific antibody may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). For example, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., LIGHT. Preferably, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen (or epitope). Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present disclosure and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” by splicing genes from a mouse antibody molecule, of appropriate antigen specificity, together with genes from a human antibody molecule of appropriate biological activity can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

Alternatively, antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications.

Additionally, standard techniques known to those of skilled in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody of the present disclosure, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.

As used herein, “Heavy Component” of an anti-ROR1/anti-4-1BB bispecific antibody of the present disclosure may comprise (1) a heavy chain of anti-ROR1 antibody and (2) a heavy chain variable region and light chain variable region of anti-4-1BB antibody when the bispecific antibody comprises an anti-ROR1 full-length antibody and an anti-4-1BB scFv; or may comprise (1) a heavy chain of anti-4-1BB antibody and (2) a heavy chain variable region and light chain variable region of anti- ROR1 antibody when the bispecific antibody comprises an anti-4-1BB full-length antibody and an anti-ROR1 scFv.

As used herein, “Light Component” of an anti-ROR1/anti-4-1BB bispecific antibody of the present disclosure may comprise: a light chain of anti-ROR1 antibody when the bispecific antibody comprises an anti-ROR1 full-length antibody and an anti-4-1BB scFv; or may comprise a light chain of anti-4-1BB antibody when the bispecific antibody comprises an anti-4-1BB full-length antibody and an anti-ROR1 scFv.

Therapeutic Use of the bispecific antibody

The bispecific antibody provided herein is capable of simultaneously bind to ROR1 protein and the 4-1BB protein on the surface of cells, thereby exhibiting improved effects in immunotherapies and/or cancer therapies, for example, by activating immune response at the tumor microenvironment. Given the ability of the bispecific antibodies of the disclosure to bind to ROR1 protein and to stimulate antigen-specific T cell responses, the disclosure also provides a composition or in vitro and in vivo methods of using the antibodies of the disclosure to stimulate, enhance or upregulate antigen-specific T cell responses.

An embodiment provides a pharmaceutical composition comprising the bispecific antibody as described above. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be used for stimulating an immune response (e.g., an antigen-specific T cell response), and/or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, such as a cancer.

Another embodiment provides a method of stimulating an immune response (e.g., an antigen-specific T cell response), and/or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, such as a cancer, in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the bispecific antibody or the pharmaceutical composition. The subject may be one in need of stimulating an immune response (e.g., an antigen-specific T cell response), and/or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, such as a cancer. The method may further step of identifying the subject in need of stimulating an immune response, or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, prior to the administering step.

Another embodiment provides a use of the bispecific antibody or the pharmaceutical composition in stimulating an immune response (e.g., an antigen-specific T cell response), and/or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, such as a cancer. Another embodiment provides a use of the bispecific antibody in preparing a medicament for stimulating an immune response (e.g., an antigen-specific T cell response), and/or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, such as a cancer.

In an embodiment, the subject may be selected from mammals including humans, monkeys, rats, mice, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on, or a cell or tissue obtained therefrom, but are not limited thereto. For example, the subject may be one in need of stimulating an immune response (e.g., an antigen-specific T cell response), and/or treating and/or preventing a disease associated with ROR1, 4-1BB, or both thereof, such as a cancer. For example, the subject may be a mammal (e.g., a human) suffering from a cancer. In other embodiment, the subject may be a cell separated (isolated) from a mammal, for example, a mammal suffering from the disease selected from cancers infectious diseases, autoimmune reactions, nervous system disorders, and the like (e.g., a cancer cell or a cell separated (isolated) from an infectious region in the mammal, or a T cell, such as a tumor-infiltrating T lymphocyte, a CD4⁺ T cell, a CD8⁺T cell, or the combination thereof). In a specific embodiment, the disease may be one associated with expression or high-expression (overexpression) of ROR1, for example, the disease may be a cancer associated with expression or high-expression (overexpression) of ROR1. For example, the "a cancer associated with high expression of ROR1" may refer to a cancer related to a cancer cell which expresses ROR1 higher than ROR1 non-expressing cancer cell (such as cancer cell line BT474, etc.).

In the pharmaceutical compositions, methods and/or uses provided herein, the disease associated with ROR1, 4-1BB, or both thereof may be one associated with activation (e.g., abnormal activation or over-activation) and/or overproduction (overexpression) of ROR1, 4-1BB, or both thereof. For example, the disease may be at least one selected from cancers (or tumors), infectious diseases (e.g., various inflammations, etc.), autoimmune reactions, nervous system disorders, and the like.

The cancer may be a solid cancer or blood cancer. The cancer may be, but not limited to, one or more selected from the group consisting of breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell carcinoma of the lung, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung), peritoneal carcinoma, skin cancer, squamous cell carcinoma, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, cervix cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, and the like. The cancer may be a primary cancer or a metastatic cancer.

As used herein, the term “prevention and/or treatment of cancer” may refer to cancer cell death, inhibition of cancer cell proliferation, alleviation of symptoms associated with cancer, inhibition of metastasis of cancer, etc.

As used herein, the term “stimulation of immune response” may refer to 4-1BB signal activation, enhancement in any immune response associated with 4-1BB, such as 4-1BB-induced signal activation (e.g., 4-1BB-induced NF-kB signal activation, increase in release of cytokine, target cell killing by immune cells, such as T cells, and the like, but not be limited thereto). In some embodiment, the enhancement of immune response by the bispecific antibody provided by this disclosure may occur be in the presence of ROR1 (under the condition of ROR expression).

The pharmaceutical compositions may comprise an effective amount of the bispecific antibody, and an acceptable carrier. In some embodiments, the composition may further comprise a second anticancer agent (e.g., an immune checkpoint inhibitor).

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, and/or excipient, in addition to the bispecific antibody as an active ingredient. In a specific embodiment, the term “pharmaceutically acceptable” may refer to approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutically acceptable carrier, diluent, and/or excipient may be anyone selected from those commonly used for the formulation of antibodies. For example, the pharmaceutically acceptable carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.

The pharmaceutical composition may further comprise one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, preservative, and the like.

The bispecific antibody or the pharmaceutical composition may be administered to the subject orally or parenterally. The parenteral administration may be intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, or rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the bispecific antibody or the compositions may be administered using an optional device that enables the active ingredient to be delivered to target cells (e.g., cancer cells).

The administration of the bispecific antibody may be conducted by one or more techniques well established in the art.

As used herein, the term “the pharmaceutically effective amount (dosage)” may refer to an amount of an active ingredient (bispecific antibody) at which the active ingredient (bispecific antibody) can exert pharmaceutically meaningful effects in preventing or treating cancer. The pharmaceutically effective amount of the bispecific antibody, or a suitable dosage of the pharmaceutical composition indicated by an amount of the bispecific antibody, may be prescribed in a variety of ways, depending on various factors, such as age, body weight, gender, pathologic conditions, diets, excretion speed, and/or reaction sensitivity of a patient, formulation types, administration time, administration route, administration manner, and the like. For example, the pharmaceutically effective amount of the bispecific antibody, or a suitable dosage of the pharmaceutical composition, may be in the range from about 0.001 to about 1000 mg(amount of the bispecific antibody)/kg(body weight), about 0.01 to about 100 mg/kg, or 0.1 to 50 mg/kg per day for an adult.

The pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier and/or excipient into a unit or a multiple dosage form by a method easily carried out by a skilled person in the pertinent art. The dosage form may be a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further comprise a dispersing or a stabilizing agent.

Diagnostic Use of the bispecific antibody

Over-expression and/or over-activation of ROR1 and/or 4-1BB is observed in a biological sample (e.g., cells, tissues, blood, serum, etc.) from a patient suffering from a certain cancer (for example, tumor cell), and/or patients having ROR1 and/or 4-1BB-over-expressing cells are likely responsive to treatments with the bispecific antibody. Accordingly, the bispecific antibody of the present disclosure can also be used for diagnostic and prognostic purposes.

An embodiment provides a pharmaceutical composition for diagnosing a disease associated with ROR1, 4-1BB, or both thereof, the composition comprising the bispecific antibody. In another embodiment, provided is a use of the bispecific antibody for diagnosing a disease associated with ROR1, 4-1BB, or both thereof.

Another embodiment provides a method of diagnosing a disease associated with ROR1, 4-1BB, or both thereof, the method comprising contacting a biological sample obtained from a patient with the bispecific antibody, and detecting antigen-antibody reaction or measuring a level of antigen-antibody reaction in the biological sample. In this method, when the antigen-antibody reaction is detected in the biological sample or the level of the antigen-antibody reaction in the biological sample is higher than that of a normal sample, the patient from whom the biological sample is obtained may be determined as a patient with a disease associated with ROR1, 4-1BB, or both thereof. Therefore, in some embodiments, the method may further comprise contacting a normal sample with the bispecific antibody, and measuring a level of an antigen-antibody reaction in the normal sample. In addition, the method may further comprise comparing the level of the antigen-antibody reaction in the biological sample and in the normal sample, after the measuring step. In addition, after the detecting step or comparing step, the method may further comprise determining the patient as a patient with a disease associated with ROR1, 4-1BB, or both thereof, when the antigen-antibody reaction is detected in the biological sample or the level of the antigen-antibody reaction in the biological sample is higher than that of the normal sample.

The disease associated with ROR1, 4-1BB, or both thereof may be one associated with activation (e.g., abnormal activation or over-activation) and/or overproduction (overexpression) of ROR1, 4-1BB, or both thereof. For example, the disease may be a cancer, as described above.

In the diagnosing composition and method, the biological sample may be at least one selected from the group consisting of a cell, a tissue, body fluid (e.g., blood, serum, lymph, etc.) and the like, obtained (separated) from a patient to be diagnosed. The normal sample may be at least one selected from the group consisting of a cell, a tissue, body fluid (e.g., blood, serum, lymph, urine, etc.) and the like, obtained (separated) from a patient having no disease associated with ROR1, 4-1BB, or both thereof. The patient may be selected from a mammal, such as a human. Upon optional pre-treatment of the sample, the sample can be incubated with the bispecific antibody of the present disclosure under conditions allowing the antibody to interact with a ROR1 and/or 4-1BB protein potentially present in the sample.

Presence and/or level (concentration) of the ROR1 and/or 4-1BB protein in the sample can be used for identifying a patient who is suitable for a treatment with the bispecific antibody, or a patient who is responsive or susceptive to the treatment with the bispecific antibody.

An embodiment provides a pharmaceutical composition identifying a patient who is suitable for a treatment with the bispecific antibody, or a patient who is responsive or susceptive to the treatment with the bispecific antibody, the composition comprising the bispecific antibody. In another embodiment, provided is a use of the bispecific antibody for identifying a patient who is suitable for a treatment with the bispecific antibody, or a patient who is responsive or susceptive to the treatment with the bispecific antibody. Another embodiment provides a method of identifying a patient who is suitable for a treatment with the bispecific antibody, or a patient who is responsive or susceptive to the treatment with the bispecific antibody, the method comprising contacting a biological sample obtained from a patient with the bispecific antibody, and detecting antigen-antibody reaction or measuring a level of antigen-antibody reaction in the biological sample.

An embodiment provides a composition for detection of ROR1, 4-1BB, or both thereof simultaneously, in a biological sample, the composition comprising the bispecific antibody. Another embodiment provides a method of detection of ROR1, 4-1BB, or both thereof simultaneously, in a biological sample, the method comprising contacting the biological sample with the bispecific antibody; and detecting (measuring) an antigen-antibody reaction (binding) between the bispecific antibody and ROR1, 4-1BB, or both thereof.

In the detecting composition and the detecting method, the term “detection of ROR1, 4-1BB, or both thereof” may refer to, but not be limited to, detection of presence (and/or absence) and/or level of ROR1, 4-1BB, or both thereof in the biological sample.

In the method of detection, when an antigen-antibody reaction is detected, it can be determined that ROR1, 4-1BB, or both thereof are present in the biological sample, and when an antigen-antibody reaction is not detected, it can be determined that ROR1, 4-1BB, or both thereof are absent (not present) in the biological sample. Therefore, the method of detection may further comprise, after the detecting step, determining that ROR1, 4-1BB, or both thereof are present in the biological sample when an antigen-antibody reaction is detected, and/or that ROR1, 4-1BB, or both thereof are absent (not present) in the biological sample, when an antigen-antibody reaction is not detected.

In the method of detection, the level of ROR1, 4-1BB, or both thereof may be determined according to the degree of the antigen-antibody reaction (e.g., the amount of antigen-antibody complex formed by the antigen-antibody reaction, the intensity of any signal obtained by the antigen-antibody reaction, and the like, which can be measured by any conventional means).

The biological sample may comprise at least one selected from the group consisting of a cell (e.g., a tumor cell), a tissue (e.g., a tumor tissue), body fluid (e.g., blood, serum, etc.), and the like, obtained or isolated from a mammal such as a human. The steps of the method of detection may be conducted in virto.

In the diagnosing method and/or detecting method, the step of detecting the antigen-antibody reaction or measuring a level of the antigen-antibody reaction may be performed by any general method known to the relevant art, such as general enzymatic reactions, fluorescent reactions, luminescent reactions, and/or detection of radiation. For example, the step may be performed by a method selected from, but not limited to, the group consisting of immunochromatography, immunohistochemistry (IHC), enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), western blotting, microarray, flow cytometry, surface plasmon resonance (SPR), and the like, but not be limited thereto.

Polynucleotides Encoding the Antibodies and Methods of Preparing the Antibodies

An embodiment provides a polynucleotide encoding the bispecific antibody. In particular, an embodiment provides a polynucleotide encoding a heavy chain of the bispecific antibody in an IgG-scFv form. Other embodiment provides a polynucleotide encoding a light chain of the bispecific antibody in the IgG-scFv form. The IgG-scFv form may refer to a kind of a bispecific antibody comprising a full-length IgG antibody targeting (binding to) one of ROR1 and 4-1BB proteins and a scFv fragment targeting (binding to) the other one, wherein the scFv is linked to a C-terminus and/or N-terminus of the full-length IgG antibody directly (without a peptide linker) or via a peptide linker.

In an embodiment, when the bispecific antibody in an IgG-scFv form comprises a full-length IgG antibody against ROR1 and a scFv fragment against 4-1BB, the polynucleotide may comprise a first polynucleotide encoding a heavy component, that is, a heavy chain of the full-length IgG antibody against ROR1 and a scFv fragment against 4-1BB that is linked to a C-terminus and/or N-terminus of the full-length IgG antibody directly or via a peptide linker; and a second polynucleotide encoding s light component, that is, a light chain of the full-length IgG antibody against ROR1.

In another embodiment, when the bispecific antibody in an IgG-scFv form comprises a full-length IgG antibody against 4-1BB and a scFv fragment against ROR1, the polynucleotide may comprise a first polynucleotide a heavy component, that is, a heavy chain of the full-length IgG antibody against 4-1BB and a scFv fragment against ROR1 that is linked to a C-terminus and/or N-terminus of the full-length IgG antibody directly or via a peptide linker; and a second polynucleotide encoding a light component, that is, a light chain of the full-length IgG antibody against 4-1BB.

Another embodiment provides a recombinant vector comprising the polynucleotide. The recombinant vector may be used as an expression vector of the polynucleotide in a host cell. For example, the recombinant vector may comprise the first polynucleotide and the second polynucleotide together in one vector or separately in two vectors. Another embodiment provides a recombinant cell comprising the first polynucleotide and the second polynucleotide. For example, the recombinant cell may be a cell transfected with the recombinant vector.

Another embodiment provides a method of preparing the bispecific antibody, comprising expressing the polynucleotide, for example the first polynucleotide and the second polynucleotide, in a cell. The step of expressing the polynucleotide may be conducted by culturing the cell comprising the polynucleotide (for example, in a recombinant vector) under a condition allowing the expression of the polynucleotide. The method may further comprise isolating and/or purifying the bispecific antibody from the cell culture, after the step of expressing or culturing.

The term “vector” refers to a means for expressing a target gene in a host cell, as exemplified by a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be constructed from plasmids frequently used in the art (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), phages (for example, λgt4λB, λ-Charon, λΔz1, and M13) or by manipulating viruses (for example, SV40, etc.).

In the recombinant vector, the polynucleotide may be operatively linked to a promoter. The term “operatively linked” is intended to pertain to a functional linkage between a nucleotide sequence of interest and an expression regulatory sequence (for example, a promoter sequence). When being “operatively linked”, the regulatory element can control the transcription and/or translation of the nucleotide of interest.

The recombinant vector may be constructed typically as a cloning vector or an expression vector. For recombinant expression vectors, a vector generally available in the relevant art for expressing a foreign protein in plant, animal, or microbial cells may be employed. Various methods well known in the art may be used for the construction of recombinant vectors.

For use in hosts, such as prokaryotic or eukaryotic cells, the recombinant vector may be constructed accordingly. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., a pLκλ promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter, etc.), a ribosomal binding site for initiating translation, and transcriptional/translational termination sequences. On the other hand, an expression vector for use in a eukaryotic host includes an origin of replication operable in a eukaryotic cell, such as an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and a BBV origin of replication, but is not limited thereto. In addition, the expression vector typically includes a promoter derived from genomes of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV), and a polyadenylation sequence as a transcription termination sequence.

The recombinant cell may be prepared by introducing the recombinant vector into a suitable host cell. As long as it allows the sequential cloning and expression of the recombinant vector in a stable manner, any host cell known in the art may be employed in the present disclosure. Examples of the prokaryotic host cell available for the present disclosure may be selected from E. coli, Bacillus spp. such as Bacillus subtilis and Bacillus thuringiensis, and enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that may be used for transformation may selected from, but are not limited to, Saccharomyce cerevisiae, insect cells, and animal cells, such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK.

The polynucleotide or a recombinant vector carrying the same may be introduced (transfected) into a host cell using a method well known in the relevant art. For example, this transfection may be carried out using a CaCl2 or electroporation method when the host cell is prokaryotic. For eukaryotic host cells, the genetic introduction may be achieved using, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle bombardment.

To select a transformed host cell, advantage may be taken of a phenotype associated with a selection marker according to methods well known in the art. For example, when the selection marker is a gene conferring resistance to a certain antibiotic, the host cells may be grown in the presence of the antibiotic in a medium to select a transformant of interest.

The present disclosure relates to bispecific antibodies, each of which comprises an antibody specific to a tumor associated antigen (TAA; ROR) and an antibody specific to 4-1BB, and uses thereof. The anti-ROR1/anti-4-1BB bispecific antibody may possess high affinities to ROR1 and/or 4-1BB, and be capable of enhancing immune response and/or treating tumor (cancer) in a mammal, with reduced liver toxicities.

FIG. 1 is the result of analysis (ELISA) of the binding capacity to the ROR1 antigen of the anti-ROR1 monoclonal phage antibody prepared according to one embodiment of the present invention. It shows that each anti-ROR1 monoclonal antibody specifically binds to the extracellular domain ROR1 antigen. In FIG. 1, BCMA-Fc is a negative control group, and it shows that each anti-ROR1 monoclonal antibody specifically binds to the ROR1 antigen only and does not bind to BCMA protein or Fc used as a tag.

FIG. 2 is the result of measurement (FACS) of the binding capacity to the ROR1 antigen of the anti-ROR1 monoclonal phage antibody prepared according to one embodiment of the present invention, and JeKo-1 cell line was used as a cell expressing ROR1 on a cell surface. It shows that each anti-ROR1 monoclonal antibody specifically binds to the ROR1 expressed on the cell surface.

FIGS. 3a and 3b are the results of analysis (ELISA) of the binding capacity to the human ROR1 antigen of the anti-ROR1 IgG antibody prepared according to one embodiment of the present invention. It shows that each antibody binds to the human ROR1 antigen concentration-dependently. The result shows that the binding capacity to ROR1 is maintained, even after modifying the monoclonal phage antibody to an IgG form.

FIG. 4 is the result of analysis (ELISA) of the binding capacity to the mouse ROR1 antigen of the anti-ROR1 IgG antibody prepared according to one embodiment of the present invention. It shows that each antibody binds to mouse ROR1 antigen concentration-dependently. Through the present experiment, it was confirmed that the anti-ROR1 antibody of the present invention had cross-reactivity to mouse ROR1. It was shown that the 2A2 antibody used as a comparison group had cross-reactivity to the mouse ROR1, but the degree of binding was relatively weak compared to the anti-ROR1 antibody of the present invention.

FIG. 5 is the result of measurement (FACS) of the binding capacity to the ROR1 antigen expressed on the cell surface of the anti-ROR1 antibody prepared according to one embodiment of the present invention, and the CHO-human ROR1 cell line, the CHO-human ROR2 and the CHO-mouse ROR1 are cell lines artificially overexpressing human ROR1, human ROR2 and mouse ROR1, respectively. It was shown that each antibody specifically bound to human ROR1 expressed on the cell surface and did not bind to human ROR2 that was a family protein. In addition, it was confirmed that the anti-ROR1 antibody of the present invention had intraspecific cross-reactivity to the mouse ROR1, by confirming that it bound to a cell line artificially overexpressing the mouse ROR1. It was shown that the 2A2 antibody used as a comparison group had cross-reactivity to the mouse ROR1, but the degree of binding was relatively weak compared to the anti-ROR1 antibody of the present invention.

FIG. 6 is the result of measurement (FACS) of the binding capacity to the ROR1 antigen expressed on the cell surface of the anti-ROR1 antibody prepared according to one embodiment of the present invention, and JeKo-1 and Mino cell line, and MCF7 cell line were used as an ROR1 expression positive cell line and an ROR1 negative cell line, respectively. It was shown that each antibody specifically bound to ROR1 expressed on the cell surface and did not bind in the MCF7 that is a cell line which does not express ROR1.

FIG. 7 is the result of measurement (FACS) of the binding capacity to the ROR1 antigen expressed on the cell surface of the anti-ROR1 antibody prepared according to one embodiment of the present invention. MC38 human ROR1 cell line in which human ROR1 was artificially overexpressed in MC38 that is a mouse colorectal cancer cell line was used. It was shown that each antibody bound to a cell line overexpressing human ROR1 concentration-dependently.

FIG. 8 is the result of measuring the binding capacity to the cell expression ROR1 antigen of the anti-ROR1 antibody prepared according to one embodiment of the present invention in various cancer cell lines (FACS). Gastric cancer cell lines (AGS, NCI-N87, MKN-28, SNU-1750, SNU-16), breast cancer cell lines (HCC1187, MDA-MB-231, MDA-MB-468, HCC70, HCC1143, BT20, HCC1806, HCC1937, BT474, MCF7), lung cancer cell lines (H460, A549, NCI-H1975, H1437, Calu-6), colorectal cancer cell lines (HCT116, DLD-1, HT29), acute lymphoblastic leukemia cell lines (697, Kasumi-2), and mantle cell lymphoma cell lines (Mino, JeKo-1), which were known as cancer forms in which ROR1 was overexpressed, was used. As the result of measurement, it was confirmed that the anti-ROR1 antibody of the present invention bound to various cancer cell lines derived from cancer forms known to be that ROR1 was overexpressed.

FIGS. 9a and 9b are the results of analyzing a cancer-inhibiting efficacy of the anti-ROR1 antibody in a mouse tumor xenograft model. It was shown that each anti-ROR1 antibody effectively inhibited the growth of cancer in a severe combined immune deficiency mouse (SCID mouse) in which a mantle cell lymphoma cell line, Jeko-1 cell line was grafted. It was shown that administered all the 5 kinds of anti-ROR1 antibodies of the present invention inhibited the growth of cancer at a statistically significant level, and the degree of cancer inhibition of each antibody clone was shown as similar. As the result of measuring weights, the weight increase pattern was observed as similar in the group administering the anti-ROR1 antibody as compared to the negative control group, human IgG1 antibody (HuIgG1). This result means that the ROR1 antibody of the present invention effectively binds to a cancer cell overexpressing ROR1, thereby inhibiting the growth of cancer, and being usefully used as a cancer therapeutic agent.

FIG. 10 is the result of analyzing the mechanism of the anti-ROR1 antibody prepared according to one embodiment of the present invention. The inhibition of the growth of cancer by the antibody may be shown by various mechanisms, for example, apoptosis induction, cancer cell division inhibition, cancer angiogenesis inhibition, and/or immunocyte activation, etc., and may show the same or different mechanism for each antibody. In FIG. 10, apoptosis was analyzed as a possible mechanism, and it was shown that the apoptosis could be induced as the antibody formed polymers as the result of treating the anti-ROR1 antibody in the cell line expressing ROR1, and the 2A2 antibody could not induce the apoptosis even despite of forming polymers.

FIG. 11 shows binding affinities of anti-4-1BB antibodies according to examples to human 4-1BB, which indicates that the anti-4-1BB antibodies can bind to human 4-1BB with high affinity.

FIG. 12 shows that the anti-4-1BB antibodies according to embodiments can efficiently bind to 4-1BB expressed on mammalian cells.

FIG. 13a shows binding affinities of bispecific antibodies according to examples to human ROR1 as measured by SACE (Single antigen capture ELISA), which indicates that the bispecific antibodies can bind to human ROR1 with high affinity.

FIG. 13b shows binding affinities of bispecific antibodies according to examples to human 4-1BB as measured by SACE (Single antigen capture ELISA), which indicates that the bispecific antibodies can bind to human 4-1BB with high affinity.

FIG. 14a shows binding affinities of bispecific antibodies according to examples to human ROR1 as measured by DACE (Dual antigen capture ELISA), which indicates that the bispecific antibodies can bind to human ROR1 with high affinity.

FIG. 14b shows binding affinities of bispecific antibodies according to examples to human 4-1BB as measured by DACE (Dual antigen capture ELISA), which indicates that the bispecific antibodies can bind to human 4-1BB with high affinity.

FIG. 15a shows 4-1BB signal activation on ROR1 high expressing cell (NCI-N87) co-cultured with bispecific antibodies according to examples, which indicates that the bispecific antibodies are capable of 4-1BB signal activation when ROR1 expression level is high.

FIG. 15b shows 4-1BB signal activation on ROR1 low expressing cell (BT474) co-cultured with bispecific antibodies according to examples, which indicates that the bispecific antibodies do not exhibit 4-1BB signal activation when ROR1 expression level is low.

FIG. 16 shows cytokine release level of MC38 cells that highly express ROR1 (lower) or not (upper) when being treated with bispecific antibodies according to examples.

FIG. 17 shows cytokine release level (upper) and cell lysis level (lower) of NCI-N87 cells when being treated with bispecific antibodies according to examples.

FIG. 18 shows secretion level of cytokines, IFN-gamma (upper) and IL-2 (lower) of ROR1-expressing NCI-N87 cells when being treated with bispecific antibodies according to examples.

FIG. 19 shows lysis level of ROR1-expressing NCI-N87 cells by bispecific antibodies according to examples.

FIG. 20 shows in vivo anti-tumor efficacies of bispecific antibodies according to examples in 4-1BB knock-in mice.

Hereafter, the present invention will be described in detail by examples.

The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

Example 1: Preparation of anti-ROR1 monoclonal antibody

1.1. Antigen

An ROR1-ECD-Fc form of protein, in which Fc was linked to a C-terminal of an extracellular domain (ECD) of human ROR1, was used as an antigen.

Specifically, a residue corresponding to the 1^st amino acid to 406^th amino acid of the ROR1 amino acid sequence represented by NCBI reference number NP_005003.2 as a protein comprising the extracellular domain of ROR1 was used for preparation of the antigen. The gene encoding the extracellular domain of ROR1 was used by purchasing cDNA of Origene company (Origene, RC214967). In addition, to purify the ROR1 extracellular domain later, a gene encoding Fc protein derived from a human IgG1 was synthesized and linked to the 3’ terminal of the gene encoding the ROR1 extracellular domain (hereinafter, named as ‘ROR1-Fc’). The gene was introduced to a pcDNA3.1 vector, and a vector encoding an ROR1-Fc nucleic acid in a mammal cell line was secured.

The ROR1-Fc was expressed by temporarily transfecting the expression vector to a HEK 293E cell and culturing it under the condition of 8% CO₂, 37℃ in a DMEM-F12 medium, and the medium was collected per 72 hours, and then mediums were combined and the Fc-ROR1 ECD protein was purified by using a protein A affinity chromatography.

1.2. Screening of full human anti-ROR1 monoclonal antibody

1.2.1. Preparation of library phage

After culturing 2x10¹⁰E. coli having a human-derived scFv (single-chain variable fragment) library (Yang et. al., 2009 Mol. Cells 27:225) gene having the binding variety to various antigens in a medium comprising 2X YT (Amresco, J902-500G), carbenicillin (Duchefa, C0109.0025) 100μg/ml, and 2% glucose (sigma, G7021) at 37℃ for 2 hours to 3 hours (OD600=0.5~0.7), a helper phage was infected and then it was cultured in a 2X YT [2X YT, carbenicillin, kanamycin (Duchfa, K0126) 70μg/ml, 1 mM IPTG(Duchefa, I1401)] medium at 30℃ for 16 hours, and thereby phage packing was induced. Then, after centrifuging the cultured cells (6000 rpm, 15min, 4℃), 4% PEG8000 (sigma, P2139) and 3% NaCl (Samchun, S2097) were added to a supernatant and it was melted well, and then it was reacted on ice for 1 hour. After centrifuging (8000rpm, 20min, 4℃) again, PBS (Phosphate buffered saline, Gibco 10010-023) was added to a pellet and it was suspended, and then centrifuged (12000rpm, 10min, 4℃), and the supernatant comprising the library phage was put into a new tube and it was stored at 4℃ before use.

1.2.2. Panning through phage display

To sort antibodies binding to a human ROR1 protein, using the ROR1-Fc protein prepared in Example 1.1, panning was progressed 3 times in total as follows.

Specifically, after adsorbing a protein on a surface of test tube at 4℃ overnight, by adding 10μg/ml concentration of ROR1-Fc and a negative control group-Fc (BMCA-Fc) in PBS into an immunotube (maxisorp 444202), bovine serum albumin (BSA) 3% solution was added to the test tube and the surface in which the ROR1-Fc was not adsorbed was protected. After emptying the test tube, the antibody phage library of 10¹²CFU dispersed in BSA 3% solution was put into the immunotube in which the control group Fc protein was adsorbed and it was reacted for 1 hour (negative selection). Then, phages which were not bound to the negative control group Fc were recovered and were combined to the immunotube in which the ROR1-Fc was adsorbed. Phages which were bound non-specifically were washed with a PBS-T (Phosphate buffered saline-0.05% Tween 20) solution 5 times ~ 30 times to remove, and the remained antigen-specific phage antibodies were recovered by using 100mM triethylamine solution. After neutralizing the recovered phages with 1M Tris buffer (pH 7.4), they were infected by ER2537 E. coli at 37℃ for 1 hour, and the infected E. coli was painted out on a 2X YT agar medium and cultured at 37℃ overnight. Next day, the cultured E. coli was suspended in a 4ml of 2X YT carbenicillin culture solution and 15% glycerol was added, and a part was stored at -80℃ and the rest was used for preparing phages for next experiments. By repeating this process 3 rounds in total, an ROR1 antigen-specific phage pool was amplified and concentrated.

1.2.3. Single clone screening

To sort monoclonal antibodies specifically binding to ROR1 from the phage pool obtained through the panning, the experiment as follows was performed.

To isolate monoclones from the concentrated pool, after smearing the phage pool on a LB-tetracycline/carbenicillin agar medium and culturing, a single colony was secured. Then, after inoculating monoclones on a 96-deep well plate in which 400μl of 2X YT-tetracycline/carbenicillin medium was put per well and cultivating overnight, 10μl culture solution was put on a new 96-deep well plate in which 390μl of 2X YT-tetracycline/carbenicillin medium was put and it was cultured at 37℃ for 4 hours. 1mM IPTG was put into the culture solution and it was cultured at 30℃ overnight. The culture solution cultured overnight was centrifuged to take a supernatant.

Then, clones expressing a soluble monoclonal scFv which binds to a human ROR1-Fc antigen were selected by using the ELISA method as follows (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1sted. ColdSpringHarborLaboratoryPress. NY. USA. pp.11.9-11.12). Specifically, the recombinant human ROR1-Fc or BCMA-Fc prepared in Example 1-1 of 100ng per well was put on a 96-well microtiter plate (Nunc-Immuno Plates, NUNC, USA) and it was coated at 4℃ overnight. The BCMA-Fc , used as a negative control group, is a recombinant protein in which the extracellular domain region of the human BCMA protein was linked to human Fc. 3% BSA of 200μL per well was put and blocking was performed at 37℃ for 2 hours. The monoclonal phage supernatant was mixed with 3% BSA at the ratio of 1:1, and this mixed solution was loaded on the well 100μL each, and then it was reacted at 37℃ for 2 hours. After washing with PBST 300μL 5 times, the anti-HA HRP binding antibody was put and it was reacted at 37℃ for 1 hour, and then it was washed with PBST 5 times. After color development by putting TMB (Tetramethylbenzidine, Sigma, T0440) 100μL, 1N H₂SO₄ 50μL was put and the reaction was stopped, and then the absorbances at 450nm and 650nm were measured, and clones of having absorbance value (450nm-650nm) of 1.0 or more when coated with ROR1 1μg/mL were selected (FIG. 1).

Then, clones binding to a cell line expressing ROR1 were screened by flow cytometry. Specifically, the monoclonal scFv supernatant 100μl was reacted with a cancer cell line overexpressing ROR1 (JeKo-1), and then it was washed with PBS twice. After reacting with the anti-HA-FITC antibody (Sigma, H7411) at 4℃ for 30 min and washing with PBS twice, it was suspended with PBS 200μl, and clones binding to Jeko-1 cell line were sorted by using FACSCalibur flow cytometer (FIG. A-2).

From them, 10 antibody clones binding to the recombinant human ROR1 protein and ROR1 expressing cell line (AB4, A2F2, A2F3, BA6, CC9, C2E3, DG6 D2B12, A2F2 M1, and BA6 M1) were sorted, and the amino acid sequence of heavy chain variable and light chain variable regions and CDR sequence of each antibody are as the following Tables 6-9.

Clone	CDR Sequences of Heavy Chain Variable (VH)						VH
	CDR1		CDR2		CDR3		VH
	Sequence	SEQ ID NO	Sequence	SEQ ID NO	Sequence	SEQ ID NO	SEQ ID NO
AB4	SYDMS		1	WISPDSGSIYYADSVKG	6	PTGRFDY	15	45
A2F2	DYYMS	2	SISPDGSNTYYADSVKG	7	NLRAFDY	16	46
A2F3	SYDMS	1	WISPGGGSKYYADSVKG	8	VNGRFDY	17	47
BA6	NYDMS		3	AIYHSGSSKYYADSVKG	9	GGNGAWDTGFDY	18	48
CC9	SYDMS		1	GISHGSGNKYYADSVKG	10	RLSLRRRPSYYSDNAMDV	19	49
C2E3	NYAMS		4	SISHNSGSTYYADSVKG	11	FISARKSLGRSYSNGMDV	20	50
DG6	DYDMS	5	VISPDGGSIYYADSVKG	12	DVVECNMNPCSYDNAMDV	21	51
D2B12	NYDMS	3	SISPSSGSSIYYADSVKG	13	APGWCQAPSCYYDNAMDV	22	52
A2F2 M1	DYYMS		2	SISPDASNTYYADSVKG	14	NLRAFDY	16	53
BA6 M1	NYDMS		3	AIYHSGSSKYYADSVKG	9	GGNAAWDTGFDY	23	54

Clone	CDR Sequences of Light Chain Variable (VL)						VL
	CDR1		CDR2		CDR3		VL
	Sequence	SEQ ID NO	Sequence	SEQ ID NO	Sequence	SEQ ID NO	SEQ ID NO
AB4	SGSSSNIGNNNVN	24	YDNKRPS	32	GTWDASLSGYV	40	55
A2F2	SGSSSNIGSNTVY	25	ANSQRPS	33	GSWDYSLSGYV	41	56
A2F3	SGSSSNIGNNNVS	26	ADSHRPS	34	ATWDYSLSGYV	42	57
BA6	SGSSSNIGSNDVS		27	YDNNRPS	35	GAWDDSLSGYV	43	58
CC9	TGSSSNIGNNAVN		28	YDSNRPS	36	GAWDDSLSGYV	43	59
C2E3	TGSSSNIGSNDVT		29	ADSKRPS	37	GTWDYSLSGYV	44	60
DG6	SGSSSNIGSNYVS	30	DDSHRPS	38	GAWDDSLSGYV	43	61
D2B12	SGSSSNIGNNDVS	31	DDSQRPS	39	GAWDDSLSGYV	43	62
A2F2 M1	SGSSSNIGSNTVY		25	ANSQRPS	33	GSWDYSLSGYV	41	63
BA6 M1	SGSSSNIGSNDVS		27	YDNNRPS	35	GAWDDSLSGYV	43	58

Heavy Chain Variable Region (VH) Sequence of anti-ROR1 antibody

Clone	Heavy Chain Variable Region (VH) Sequence	SEQ ID NO
AB4	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISPDSGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPTGRFDYWGQGTLVTVSS		45
A2F2	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSS	46
A2F3	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISPGGGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVNGRFDYWGQGTLVTVSS	47
BA6	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSS	48
CC9	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSGISHGSGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRLSLRRRPSYYSDNAMDVWGQGTLVTVSS	49
C2E3	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISHNSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFISARKSLGRSYSNGMDVWGQGTLVTVSS		50
DG6	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYDMSWVRQAPGKGLEWVSVISPDGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVVECNMNPCSYDNAMDVWGQGTLVTVSS	51
D2B12	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSSISPSSGSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGWCQAPSCYYDNAMDVWGQGTLVTVSS	52
A2F2 M1	EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDASNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSS	53
BA6 M1	EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNAAWDTGFDYWGQGTLVTVSS	54

Light Chain Variable Region (VL) Sequence of anti-ROR1 antibody

Clone	Light Chain Variable Region (VL) Sequence	SEQ ID NO
AB4	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNNVNWYQQLPGTAPKLLIYYDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDASLSGYVFGGGTKLTVLG		55
A2F2	QSVLTQPPPASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG	56
A2F3	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNNVSWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVLG	57
BA6/ BA6 M1	QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	58
CC9	QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNAVNWYQQLPGTAPKLLIYYDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	59
C2E3	QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNDVTWYQQLPGTAPKLLIYADSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDYSLSGYVFGGGTKLTVLG		60
DG6	QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLIYDDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	61
D2B12	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVSWYQQLPGTAPKLLIYDDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	62
A2F2 M1	QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG	63

Constant Region

	Amino Acid Sequence	SEQ ID NO
Heavy Chain Constant Region (WT)	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	89
Heavy Chain Constant Region (NA)	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	90
Light Chain Constant Region	QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS	91

The nucleic acid sequences encoding the variable regions and CDR sequences are comprised as part of nucleic acid sequences encoding a full-length heavy chain and full-length light chain which comprise the above constant region in addition to the variable region as described above.

1.3. Conversion of anti-ROR1 scFv into total IgG form and production thereof

1.3.1. Cloning of anti-ROR1 scFv into full IgG form

To convert the sequence of each ROR1-specific monoclonal phage antibody, secured in Example 1, into a full IgG form, a nucleic acid encoding heavy chain and light chain variable regions of each clone secured in Example 1 was synthesized (Genotech, Korea). A gene encoding a human IgG1 subtype of heavy chain and light chain constant regions (SEQ ID NO: 89 and 91, respectively) protein was synthesized and was linked to the nucleic acid encoding each heavy chain and light chain variable region. Each of the nucleic acid encoding light chain and heavy chain of each antibody was cloned into a pcDNA3.1-based expression vector, and a vector encoding the nucleic acid for the antibody in a CH0-S mammal cell line was secured.

A chimera antibody in which a human IgG1 was linked to the variable region of 2A2 (US 9,316,646) that was the conventional anti-ROR1 antibody was used as a comparison group antibody.

1.3.2. Expression of anti-ROR1 IgG antibody

CHO-S cells were cultured at 8% CO₂, 37℃ for 1 day in a CD-CHO (Gibco, 10743) medium at a concentration of 1.5x10⁶ cells/ml. After preparing cells grown as 2.5~3x10⁶ cells/ml at a concentration of 2.1x10⁶ cells/ml using the CD-CHO medium comprising 1% DMSO on the day of DNA transfection, they were cultured at 8% CO₂, 37℃ for 3 hours. After centrifuging at 3000 rpm for 15 min and having the supernatant removed, it was resuspended in a RPMI 1640 medium comprising 2.5% FBS. Then, each vector expressing a heavy chain and a light chain as obtained from Example 2-1 was diluted in Opti-MEM medium, in 1μg per medium ml, and 8μg per culture medium ml of PEI (Polysciences, 23966, stock concentration: 1mg/ml) was diluted. After the vector and PEI mixture were mixed and stood at a room temperature for 10 min, it was put into a flask comprising the cell prepared as above, and then it was cultured at 5% CO₂, 37℃, 100 rpm for 4 hours, and the same volume of CD-CHO medium as the culturing volume was put, and then it was cultured at 8% CO2, 37℃, 110 rpm for 4 days.

1.3.3. Separate purification of anti-ROR1 IgG antibody

After passing an equilibrium buffer solution (50 mM Tris-HCl, pH7.5, 100 mM NaCl) through into Mab selectsure (GE healthcare, 5mL) and thereby equilibrating, the culture solution of Example 1.3.2 was passed through into a column (Mab selectsure (GE healthcare, 5mL)) in order to allow the expressed antibody to bind to the column. Then, after eluting it with 50mM Na-citrate (pH 3.4), 100 mM NaCl solution, it was neutralized by using 1M Tris-HCl (pH 9.0) so that the final pH was 7.2. The buffer solution was exchanged to PBS (phosphate buffered saline, pH 7.4).

1.4. Analysis of binding specificity to ROR1 of anti-ROR1 IgG antibody

1.4.1. Analysis of binding capacity to ROR1 antigen (extracellular domain) of anti-ROR1 IgG antibody (ELISA)

The specific binding capacity to the antigen of IgG antibody of each clone selected and prepared in Examples 1.1 and 1.3 was analyzed as follows.

The anti-ROR1 antibody-antigen binding affinity was estimated by using an ELISA-based solution binding test. Specifically, a 96-well microtiter plate (Nunc-Immuno Plates, NUNC) was coated with the ROR1 protein at a concentration of 1μg/ml in a PBS solution as described below at 4℃ for 16 hours, and non-specific binding sites were blocked with 3% BSA (bovine serum albumin) for 2 hours. For this, as the ROR1 protein, in case of human ROR1, ROR1-Fc of Example 1.1 or recombinant ROR1-His (Sino Biological, 13968-H08H) was used. The ROR1-His used for ELISA was a protein of sino biological company (13968-H08H) as described in the above sentence, and ROR1-His of Example 1 or recombinant mouse ROR1 protein was used (Acrobiosystems, RO1-M5221-100μg).

Then, after adding the anti-ROR1 antibody prepared in Example 1.3 at the concentration described in FIG. 2 into the microtiter plate on a 96-well microtiter plate, the binding capacity was analyzed with ELISA as follows. Specifically, after constant temperature treatment for 2 hours, the plate was washed with PBS comprising 0.05% tween 20 5 times, and then a HRP-conjugated Fab multiclonal antibody reagent (Pierce, 31414) was diluted at 1:10,000 ratio, and was put into the washed microtiter plate, and it was incubated at 37℃ for 1 hour, and the anti-ROR1 antibody bound to the plate was detected. After reaction, it was color-developed by using TMB (Tetramethylbenzidine, Sigma, T0440). The enzymatic reaction was stopped by 0.5mol/L of sulfuric acid, and the absorbances at 450nm and 650nm were measured by using a micro plate reader device (molecular device) (450nm-650nm).

The result was disclosed in FIGS. 3a and 3b, and FIG. 4, and it was confirmed that the anti-ROR1 antibody of the present invention bind to the human ROR1 and mouse ROR1 in a concentration dependent manner. In addition, when comparing the cross-reactivity to the mouse ROR1 protein, it was shown that the anti-ROR1 antibody of the present invention had excellent binding capacity as compared to 2A2 comparison antibody used as the comparison group.

1.4.2. Measurement of specific binding capacity to cell surface-expressed ROR1 antigen of anti-ROR1 IgG antibody (FACS)

Binding to a cell surface-expressed antigen is a necessary requisite for an antibody against a specific antigen to be used in a living body as an antibody for treatment, etc. In case of some antibodies, they bind to purified antigens but do not bind to a cell surface-expressed antigen. In such case, binding to an antigen doesn’t occur when the antibody is administered to a living body, the antibody cannot bind to a cell expressing the antigen, and therefore, it cannot show the activity in a living body such as an antibody for treatment.

Accordingly, whether the anti-ROR1 antibody of the present invention binds to a cell surface-expressed ROR1 was confirmed by FACS analysis.

For this experiment, the degree of binding of the anti-ROR1 antibody and ROR1 was measured by using FACSCalibur (BD Biosciences) device with the cell lines as follows, the cell lines artificially overexpressing the ROR1 protein by transfecting an ROR1 gene temporarily (CHO-human ROR1, CHO-human ROR2, CHO-mouse ROR1) or stably (MC38-human ROR1) (FIG. 5 and FIG. 7, respectively) or the cell lines expressing ROR1 (JeKo-1, Mino) (FIG. 6) or a cell line not expressing ROR1 (MCF7) (FIG. 6).. MCF7 is a negative control group which does not express ROR1, and CHO-human ROR2 is a negative control group expressing human ROR2. JeKo-1, Mino, CHO-human ROR1, CHO-mouse ROR1 and MC38-human ROR1 are all cell lines expressing human ROR1 or mouse ROR1.

Specifically, after disassociating each cell line and washing in PBS, the number of cells was counted and set as 2x10⁵ cells/200μl PBS, and then each ROR1 monoclonal antibody in Example 3 was prepared in 10μg/mL or as 5 fold-diluted from 10μg/mL, and was reacted at 4℃ for 1 hour. After reaction, cells were washed in PBS and then the FITC-labeled constant region (Fc)-specific antibody (Goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 2.0mg/ml) was suspended in 2μl/1x105 cells/200μl PBS, and it was reacted at 4℃ for 1 hours. The confirmation of expression degree of human ROR1, human ROR2 and mouse ROR1 that were temporarily overexpressed was analyzed by using a commercially available antibody for FACS analysis (anti-ROR1: R&D Systems, FAB2000G, anti-ROR2: R&D, FAB20641P). After reaction, cells were washed in PBS and decoded by using a FACSCalibur device. The negative control group (2nd Ab) was treated only with the FITC-labeled constant region (Fc)-specific antibody. The result for the peak shift in the experimental group treated with each ROR1 monoclonal antibody was compared to the result for the shift in the control group (Mean Fluorescence Intensity Ratio,　MFI Ratio: MFI of anti-ROR1 / MFI of 2nd Ab).

The result was disclosed in FIG. 5, FIG. 6 and FIG. 7. As a result, it was confirmed that the anti-ROR1 antibody of the present invention specifically binds to the extracellular domain of human ROR1 as originally expressed in cells (FIG. 6) and human ROR1 as artificially overexpressed in cells (FIG. 5, FIG. 7), in a concentration-dependent pattern. In addition, it was confirmed that it does not bind to a family protein, human ROR2, and it has cross-reactivity to mouse ROR1 (FIG. 5). Comparing the cross-reactivity to mouse ROR1 expressed on a cell surface, it was confirmed that the degree of binding of the anti-ROR1 antibody of the present invention was more excellent as compared to the antibody used as the comparison group, 2A2 (FIG. 5).

1.4.3. Measurement of binding capacity to cell surface-expressed ROR1 antigen of anti-ROR1 IgG antibody in various cancer forms

Subsequently, whether the anti-ROR1 antibody of the present invention binds to a cell surface-expressed ROR1 in various kinds of cancer cell lines was confirmed through FACS analysis. ROR1 is expressed in various cancer cells, and it has been reported that it is overexpressed in various solid cancers such as breast cancer, renal cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer, etc. as well as hematologic malignancy such as chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), Diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL), etc.

For this experiment, various kinds of cancer cell lines were used as follows: AGS (ATCC^® CRL-1739™, human gastric adenocarcinoma), NCI-N87 (ATCC^® CRL-5822™, human gastric carcinoma), MKN-28 (KCLB 80102, human gastic adenocarcinoma), SNU-1750 (KCLB 01750, human gastric adenocarcinoma), SNU-16 (ATCC^®CRL-5974™, human gastric carcinoma), HCC1187 (ATCC^® CRL-2322™, human breast cancer TNM stage IIA grade 3), MDA-MB-231 ATCC^® HTB-26™, human breast cancer), MDA-MB-468 (ATCC^®HTB-132™, human breast cancer), HCC70(ATCC^®CRL-2315™, human breast cancer TNM stage IIIA, grade 3), HCC1143 (ATCC^®CRL-2321™, TNMstageIIA,grade3,primaryductalcarcinoma), BT20 (ATCC^® HTB-19™ human breast cancer), HCC1806 (ATCC^® CRL-2335™, human breast cancer TNM stage IIB grade 2), HCC1937 (ATCC^®CRL2336™, TNMstageIIB,grade3, primaryductalcarcinoma), BT474 (ATCC^® HTB-20™, ductal carcinoma), MCF7 (ATCC^® HTB-22™, breast cancer metastatic site), H460 (ATCC^® HTB-177™, large cell lung cancer), A549 (ATCC^® CCL-185™, lung carcinoma), NCI-H1975 (ATCC^® CRL-5908™, non-small cell lung cancer), H1437 (ATCC^® CRL-5872™, stage1,adenocarcinomanon-smallcelllungcancer), Calu-6 (ATCC^® HTB-56™, anaplastic lung carcinoma), HCT116 (ATCC^® CCL-247™, colorectal carcinoma), DLD-1 (ATCC^® CCL-221™, Dukes'type C, colorectal adenocarcinoma), HT29 (ATCC^® HTB-38™, colorectal adenocarcinoma), 697 (DSMZ ACC 42, acute myeloblastic leukemia), Kasumi-2 (ATCC^® CRL-2724™,　 acute myeloblastic leukemia), Mino (ATCC^®CRL-3000™, MantleCellLymphoma), JeKo-1 (ATCC^®CRL-3006™, MantleCellLymphoma), Jurkat (ATCC^® TIB-152™, acute T cell leukemia). For the cell lines, the binding to ROR1 was analyzed with FACS (FACSCalibur, BD Biosciences) by using the anti-ROR1 antibody of the present invention.

Specifically, after disassociating each cell line and washing in PBS, the number of cells was counted and set as 2x10⁵ cells/200μl PBS, and then the clone C2E3 among ROR1 monoclonal antibodies prepared in Example 3 was treated in 10μg/mL, and it was reacted at 4℃ for 1 hour. After reaction, cells were washed in PBS, and then the FITC-labeled constant region (Fc)-specific antibody (Goat anti-human IgG FITC conjugate, Fc specific, Sigma, F9512, concentration: 2.0mg/ml) was suspended in 2μl/1x10⁵ cells/200μl PBS, and it was reacted at 4℃ for 1 hour. After reaction, cells were washed in PBS, it was analyzed using a FACSCalibur device. The negative control group was treated only with the FITC-labeled constant region (Fc)-specific antibody. To compare the expression degrees of ROR1 among the cancer cell lines, the value of the result for the peak shift in the experimental group treated with the ROR1 monoclonal antibody (C2E3) of the present invention was divided by the result for the peak shift in the control group (Mean Fluorescence Intensity Ratio　MFI Ratio: MFI of anti-ROR1 / MFI of 2nd Ab).

The result was described in FIG. 8. As a result, it was confirmed that the anti-ROR1 antibody of the present invention binds to ROR1 expressed in various cancer cell lines derived from gastric cancer, breast cancer, lung cancer, colorectal cancer, acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL).

1.5. Measurement of affinity to ROR1 of anti-ROR1 IgG antibody

A 96 well black microplate (greiner bio one) in a biosensor tray case was installed, and 200μl of 10X KB or D.W was put in 8 wells each. 8 sensors, Anti Penta His biosensors or AR2G biosensors (ForteBio, USA), were placed for 10 min for hydration. 600μl of Analytic samples were diluted 2 folds or 3 folds to desired concentrations. It was diluted using 1X KB or 10X KB to reach a concentration of 30 ~ 0.021nM. For antigen fixation, 1μg/mL of a recombinant ROR1-His (Sino Biological, 13968-H08H) was diluted with 10X KB or Sodium Acetate pH5 buffer. For fixation, threshold was set to be 0.3nmat the loading step. The experiment was performed for 3min~10min of association and for 20min of dissociation. The prepared buffer was put into a new 96 well black microplate in accordance with octet program template in order. 10X KB or D.W 200μl used as Baseline1 was put. 200μl of the antigen ROR1-His protein (1μg/mL) was loaded. 10X KB or D.W 200μl used as Baseline2 was put. 200μl of 10X KB buffer or 1X KB corresponding the diluted antibody in 30 ~ 0.021nM, Reference blank was put into each well. The temperature of the experimental plate was fixed at 30℃. After placing all samples, the device was activated. After the experiment was finished, the result was uploaded on Octet Analysis 9.0 software, and then KD value was calculated using 1:1 fitting, and the result was described in the following Table 11. By the KD values from Octet analysis method, it was confirmed that the anti-ROR1 antibody had strong binding capacity to the ROR1 antigen.

Measurement of affinity to ROR1 of anti-ROR1 IgG antibody

NO	Clone	KD (M)	kon(1/Msec)	koff(1/sec)	Chi	R^2
1	AB4	6.39E-11	1.81E+06	1.16E-04	0.016	0.996
2	A2F2	7.73E-11	1.53E+06	1.19E-04	0.106	0.997
3	A2F3	2.40E-11	1.38E+06	3.31E-05	0.131	0.992
4	BA6	9.37E-11	2.47E+06	2.31E-04	0.138	0.993
5	C2E3	4.24E-10	5.46E+06	2.31E-03	0.176	0.966
6	CC9	7.54E-10	7.31E+05	5.51E-04	0.200	0.992
7	DG6	1.52E-10	2.23E+06	3.38E-04	0.506	0.964
8	D2B12	8.03E-10	4.50E+05	3.61E-04	0.341	0.952

1.6. Analysis of efficacy of inhibiting growth of cancer of anti- ROR1 IgG antibody in mouse tumor xenograft model

JeKo-1 cell line, a human mantle cell lymphoma which expresses ROR1, of 1 x 10⁷ cells/head was grafted into the severe combined immunodeficiency mouse (SCID), in order to construct a human cancer graft tumor mouse. After the graft, when the size of tumor reached the average 170 mm³(Day 1), group separation was conducted, and the 5 kinds of anti-ROR1 antibodies were administered at 10mg/kg twice a week, 5 times in total, by using a 1mL syringe into a mouse intraperitoneally (

Day

1, 4, 7, 10 and 14). For the negative control group, a human IgG1 having the similar structure to the anti-ROR1 antibody (InVivoPlus human IgG1 isotype control, BioXCell, BP0297) was administered at 10mg/kg twice a week, 5 times in total, intraperitoneally. The size and weight of tumor grafted into the mouse were measured just before the initial administration (Day 1), then measured just before the administration on each administration day, and measured 2 days after the final administration (Day 16).

The result was described in FIG. 9a and FIG. 9b. It was shown that the anti-ROR1 antibody inhibited the growth of cancer. The effect of growth inhibition of cancer (% Tumor Growth Inhibition, %TGI) compared to the negative control group, human IgG1 antibody (HuIgG1) was C2E3 36.0%, A2F2 28.9%, AB4 36.1%, BA6 31.7% and CC9 29.4%, on the experiment termination date (Day 16). It was shown that the anti-ROR1 antibody had a statistically significant difference compared to the HuIgG administration group (one-way layout analysis of variance, P value < 0.05) (FIG. 9a). The measurement of weight showed no significant differences among administration groups (FIG. 9b). As described in Examples 1.4.1 and 1.4.2, the anti-ROR1 antibody of the present invention had the cross-reactivity to the mouse ROR1 antigen. Therefore, the administered anti-ROR1 antibody of the present invention can bind to the mouse ROR1 which the mouse expresses by itself in addition to the human ROR1 which the xenografted human mantle cell lymphoma, JeKo-1 cell line expresses. The patterns in which weight increase between the negative control group (HuIgG1) and the anti-ROR1 antibody of the present invention were similar each other. This means that the toxicity was not induced by the anti-ROR1 antibody of the present invention. The result shows that the antibody can be usefully used as a cancer therapeutic agent.

It was shown that all the 5 kinds of anti-ROR1 antibodies of the present invention inhibited the growth of cancer in the mouse tumor xenograft model. It was shown that some antibodies (C2E3, AB4) could induce apoptosis of the ROR1 overexpressing cancer cell line, through multimerization by the anti-human Fc antibody, as described in the following Example 1.7 and FIG. 10. Since the inhibition mechanism of cancer can be possible through various mechanisms such as apoptosis induction, cancer cell division and growth inhibition, cancer angiogenesis inhibition, immunocyte activation, etc., the result of FIG. 10 described later means that the anti-ROR1 antibodies can inhibit the growth of cancer in a living body by different mechanisms for each antibody.

1.7. Analysis of apoptosis-inducing capacity of anti-ROR1 IgG antibody

To analyze a possible mechanism of the antibody showing the tumor-inhibiting capacity as Example 1.6, the apoptosis-inducing capacity was analyzed.

For this, a ROR1 high expressing cell line, JeKo-1 was centrifuged, and the medium comprising serum was removed. It was washed once using PBS, and then 5x10⁶ cells per well were seeded on a 6 well plate using RPMI1640 medium having no serum. After putting 100μg/mL of the anti-ROR1 antibody of the present invention and 300μg/ml of the anti-human Fc antibody (Thermo Fisher, 31125) in a equivalent tube at a ratio of 1:1, it was reacted at a room temperature for 10 mins, so that the cross-linked anti-ROR1 antibody forms through the anti-human Fc antibody. When 150μl of the mixture was put into each well in which 1.5ml of medium was included, so that the final amount of the antibody treated was to be 10μg/mL for the anti-ROR1 antibody and 30μg/mL for the anti-human Fc antibody. Then it was cultured under the condition of 5% CO2, 37℃ for 24 hours and was reacted.

Then, to confirm whether the treatment of anti-ROR1 antibody and the cross-linked anti-ROR1 antibody have apoptosis capacity, cells from each well were collected and washed once with PBS. Afterward, Annexin V, a label of apoptosis, and PI, a label of cell death, were reacted on each group and the dyed degree was confirmed through FACS analysis.

The result was described in FIG. 10. It was confirmed that the apoptosis was not observed for the anti-ROR1 antibody treatment alone, but in case of some cross-linked anti-ROR1 antibody clones (C2E3, AB4) forming through the anti-human Fc antibody, the dyed degree of Annexin V and PI was increased as compared to the control group. In particular, it was confirmed that the dyed degree of the apoptosis capacity label, Annexin V, 23% and 10% were increased for C2E3 clone and AB4 clone, compared to the control group. To confirm if such an apoptosis capacity is a specific reaction induced by ROR1, the Annexin V and PI dyed degree was confirmed by the same method on non-ROR1-expressing cell lines, U266 using C2E3 clone, and it was confirmed that the apoptosis capacity was not induced for the ROR1 non-expressing cell line, U266. This demonstrates that the apoptosis capacity by the cross-linked anti-ROR1 antibody was a specific reaction associated with ROR1. As the cross-linked antibody can be formed by binding to an Fc gamma receptor through an Fc region in a living body, the cross-linked anti-ROR1 antibody with the anti-human Fc antibody can be considered to be a similar condition to in vivo phenomena. The result of analysis is one mechanism, and it means that the apoptosis can be induced in a cancer cell line overexpressing ROR1 by the anti-ROR1 antibody.

For reference, the apoptosis induction of the cancer cell line by cross-linked anti-ROR1 antibody is not a phenomenon shown in all the kinds of anti-ROR1 antibodies. For example, BA6 clone among the anti-ROR1 antibodies of the present invention and 2A2 that was the antibody used as the comparison group did not induce apoptosis of the ROR1 overexpressing cancer cell line. This means that the anti-ROR1 antibodies had the inhibiting capacity of cancer cell by different mechanisms each other. Such a difference is not limited to this theory, but may result from the dissimilarity of epitopes to which each anti-ROR1 antibody binds.

Example 2. Preparation of anti-4-1BB monoclonal antibody

2.1. Screening of full human monoclonal antibodies against 4-1BB

For panning of the library against target molecules, four rounds of panning were carried out in total using 4-1BB coated immunotubes.

Bacterial colonies from the 3 rounds of panning output were grown in SB-Carbenicilin in 96 deepwell plate until turbid, at which point 1011 pfu of VCSM13 helper phage was added to each well. After 1 h infection at 37°C with gentle shaking (80 rpm), 70 μg/mL of kanamycin was added and the cells were cultured overnight at 30°C with shaking at 200 rpm.

Next day, the plates were centrifuged and the supernatants containing the phages were added to 4-1BB antigen-coated ELISA plates blocked with 3% BSA in PBST. After 1 h incubation at room temperature, the plates were washed three times with PBST and anti M13 antibody was added. The plates were incubated for 1 h, washed three times with PBST, and the binding activity was measured using tetramethylbenzidine (TMB).

The 4-1BB specific binders were amplified for plasmid DNA sequencing. Ig light chain V genes (VL) and VH sequences were analyzed to identify unique sequences and determine sequence diversity.

Heavy chain variable regions

Antibody	VH	SEQ ID NO:
1A10	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGQRNSMREFDYWGQGTLVTVSS	98
1A10M4	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	99
1A10M11	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS		100
1A10M12	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	99
1A10M13	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS		100
1A12	EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKGLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	101
1A12M1	EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKGLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS
	102

Heavy Chain CDRs

Antibody	CDR H1	SEQ ID NO:	CDR H2	SEQ ID NO:	CDR H3	SEQ ID NO:
1A10	SYDMS	68	WISYSGGSIYYADSVKG	70	DGQRNSMREFDY	72
1A10M4	SYDMS	68	WISYSGGSIYYADSVKG	70	DAQRNSMREFDY	74
1A10M11	SYDMS	68	WISYSGGSIYYADSVKG	70	DAQRQSMREFDY	75
1A10M12	SYDMS	68	WISYSGGSIYYADSVKG	70	DAQRNSMREFDY	74
1A10M13	SYDMS	68	WISYSGGSIYYADSVKG	70	DAQRQSMREFDY	75
1A12	GYDMS	69	VIYPDDGNTYYADSVKG	71	HGGQKPTTKSSSAYGMDG	73
1A12M1	GYDMS	69	VIYPDDGNTYYADSVKG	71	HGGQKPTTKSSSAYGMDG	73

Light chain variable regions

Antibody	VL	SEQ ID NO:
1A10	QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	103
1A10M4	QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	103
1A10M11	QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	103
1A10M12	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	104
1A10M13	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	104
1A12	QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	103
1A12M1	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL	104

Light Chain CDRs

Antibody	CDR L1	SEQ ID NO:	CDR L2	SEQ ID NO:	CDR L3	SEQ ID NO:
1A10	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78
1A10M4	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78
1A10M11	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78
1A10M12	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78
1A10M13	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78
1A12	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78
1A12M1	SGSSSNIGNNYVT	76	ADSHRPS	77	ATWDYSLSGYV	78

2.2. Antigen Binding Abilities of Anti-4- 1BB Antibodies to human 4-1BB

2.2.1. Antigen binding measured by ELISA

To evaluate the antigen binding activity, the antibody candidates were subjected to ELISA test. Briefly, microtiter plates were coated with human 4-1BB-Fc protein at 0.1μg/ml in PBS, 100 μl/well at 4°C overnight, then blocked with 100 μl/well of 5% BSA. Five-fold dilutions of humanized antibodies {1A10 and 1A12} starting from 10 μg/ml were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubate with goat-anti-human IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 1 hour at RT. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630nm. As shown in FIG. 11, the anti-4-1BB antibodies tested show 4-1BB binding abilities.

2.2.2. Cell binding measured by FACS

To evaluate the antigen binding property, the antibody candidates were analyzed for its binding to mammalian expressed 4-1BB by FACS. Briefly, 4-1BB-Jurkat cells were incubated with antibodies (1A10 and 1A12). After wash by FACS buffer (1%BSA in PBS), the FITC-anti-human IgG antibody was added to each well and incubated at 4°C for 1 hour. The MFI of FITC was evaluated by FACS Caliber. As shown in FIG. 12, the anti-4-1BB antibodies tested show binding abilities to 4-1BB which expressed on cell surface and can efficiently bind to 4-1BB expressed on mammalian cells.

2.2.3. Protein kinetic for 4-1BB

To explore the binding kinetics of the humanized antibody, this example performed the affinity ranking by using Octet Red 96. As shown in Table 16 below, the anti-4-1BB antibodies tested show high 4-1BB binding affinities.

Antibody	KD (M)	kon(1/Ms)	kdis(1/s)	chi	R2
1A10	1.80E-10	6.58E+05	1.19E-04	0.0392	0.9987
1A12	1.01E-09	5.95E+05	6.03E-04	0.0525	0.9973
1E7	7.90E-10	7.55E+05	5.97E-04	0.1213	0.9951

Example 3. Preparation of anti- ROR1 /anti- 4- 1BB bispecific antibodies

A2F2M1 and BA6 clones among the ROR1 clones prepared in Example 1, and 1A10M12 and 1A12M1 clones among the anti-4-1BB clones prepared in Example 2 were exemplarily selected, to prepare anti-ROR1/anti-4-1BB bispecific antibodies in a full-length IgG X scFv form. When ROR1 is placed in full IgG part, IgG1 with ADCC reduced mutant backbone (N297A mutation; US Patent. No. 7332581, 8219149, etc.; hereinafter, “NA”) was used, and when 4-1BB is placed in full IgG part, IgG4 was used.

For example, anti-4-1BB scFv antibodies with a structure of (N’)-VL-linker-VH-(C’) were prepared using the variable regions of the full human monoclonal antibodies against 4-1BB shown in Tables 10 and 12 of Example 2.1, wherein the amino acid residue “G” at the position 44 of a heavy chain variable region was substituted with “C”, and the amino acid residue “G” at the position 103 of a light chain variable region was substituted with “C”. Such amino acid substitution from “G” to “C” in scFv can contribute to increase in stabilities of bispecific antibodies comprising the scFv as one target-specific moiety. Such mutated heavy chain variable regions and light chain variable regions of anti-4-1BB antibody for constructing an anti-4-1BB scFv are listed in Tables 17 and 18:

Heavy chain variable regions having G→C substitution (in bold) at the position 44

Antibody	VH	SEQ ID NO:
1A10	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGQRNSMREFDYWGQGTLVTVSS	79
1A12	EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS		80
1A10M4/1A10M12	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	81
1A10M11/1A10M13	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS	82
1A12M1	EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	84

Light chain variable regions having G→C substitution (in bold) at the position 103

Antibody	VL	SEQ ID NO:
1A10/1A10M4/1A10M11/1A12	QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL		85
1A10M12/1A10M13/1A12M1	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	86

A DNA segment 1 having a nucleotide sequence encoding a heavy chain of an IgG antibody of the anti-ROR1/anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1), and a DNA segment 2 having a nucleotide sequence encoding a light chain of an IgG antibody of the anti-ROR1/anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 2). Thereafter, a DNA segment 3 encoding a scFv was fused at a part of the DNA segment 1 corresponding to the c-terminus of the Fc region of the IgG antibody inserted into the plasmid 1, using a DNA segment 4 encoding a linker peptide having 18 amino acid lengths consisting of (GS)9, to construct vectors for the expression of bispecific antibodies. Furthermore, in order to stabilize scFv, additional modification was applied to generate disulfide bridge fusing VL103-VH44 to C-terminus of light chain and C-terminus of heavy chain, respectively.The sequences of the heavy chain, light chain, and scFv of the anti-ROR1/anti-4-1BB bispecific antibodies are summarized in Tables 19-22:

Bispecific antibody comprising the anti-ROR1 clone in IgG form and the anti-4-1BB clone in scFv form (ROR1x4-1BB) [BA6(NA) x 1A10 M12]

Description of Components		Amino acid sequence ((N->C))	SEQ ID NO
Heavy component
Heavy chain of anti-ROR1 antibody [ROR1-VH(B6)-(NA)]		EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	64
Linker [(GS)9]		GSGSGSGSGSGSGSGSGS	92
anti-4-1BB scFv	1A10 M12 VL	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	86
	Linker [(G4S)4]	GGGGSGGGGSGGGGSGGGGS	93
	1A10 M12 VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	81
	scFv	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	87
Full-length of Heavy component		EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	94
Light component
ROR1-VL		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	58
Light chain of anti-ROR1		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS	66

Bispecific antibody comprising the anti-ROR1 clone in IgG form and the anti-4-1BB clone in scFv form (ROR1x4-1BB) [BA6(NA) x 1A12 M1]

Description of Components		Amino acid sequence (N->C)	SEQ ID NO
Heavy component
Heavy chain of anti-ROR1 antibody [ROR1-VH(B6)-(NA)]		EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	64
Linker [(GS)9]		GSGSGSGSGSGSGSGSGS	92
anti-4-1BB scFv	1A12 M1 VL	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	86
	Linker [(G4S)4]	GGGGSGGGGSGGGGSGGGGS	93
	1A12 M1 VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	84
	scFv	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	88
Full-length of Heavy component		EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	95
Light component
ROR1-VL		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLG	58
Light chain of anti-ROR1		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS	66

Bispecific antibody comprising the anti-ROR1 clone in IgG form and the anti-4-1BB clone in scFv form (ROR1x4-1BB) [A2F2M1(NA) x 1A10 M12]

Description of Components		Amino acid sequence (N->C)	SEQ ID NO
Heavy component
Heavy chain of anti-ROR1 antibody [ROR1-VH(B6)-(NA)]		EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDASNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	65
Linker [(GS)9]		GSGSGSGSGSGSGSGSGS	92
anti-4-1BB scFv	1A10 M12 VL	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	86
	Linker [(G4S)4]	GGGGSGGGGSGGGGSGGGGS	93
	1A10 M12 VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	81
	scFv	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	87
Full-length of Heavy component		EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDASNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	96
Light component
ROR1-VL		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG	63
Light chain of anti-ROR1		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS	67

Bispecific antibody comprising the anti-ROR1 clone in IgG form and the anti-4-1BB clone in scFv form (ROR1x4-1BB) [A2F2M1(NA) x 1A12 M1]

Description of Components		Amino acid sequence (N->C)	SEQ ID NO
Heavy component
Heavy chain of anti-ROR1 antibody [ROR1-VH(B6)-(NA)]		EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDASNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	65
Linker [(GS)9]		GSGSGSGSGSGSGSGSGS	92
anti-4-1BB scFv	1A12 M1 VL	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	86
	Linker [(G4S)4]	GGGGSGGGGSGGGGSGGGGS	93
	1A12 M1 VH	EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	84
	scFv	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	88
Full-length of Heavy component		EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSSISPDASNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNLRAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS	97
Light component
ROR1-VL		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLG	63
Light chain of anti-ROR1		QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVYWYQQLPGTAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS	67

The constructed vectors were transiently expressed in ExpiCHO-S™ cells (Thermo Fisher, A29127) using (ExpiFectamine™CHO Kit, Thermo, A29129), cultured in ExpiCHO™ Expression medium (Thermo, A29100-01) under the conditions of 30 to 37℃ for 7 to 15 days in a CO2 incubator equipped with rotating shaker. Plasmid DNA (250 μg) and ExpiFectamin CHO Reagent (800 μL) were mixed with Opti-MEM® I medium (20 mL final volume) and allowed to stand at room temperature for 5 min. The mixed solution was added to 6 x 10⁶ ExpiCHO cells cultured in ExpiCHO Expression Medium and gently mixed in a shaker incubator at 37°C with a humidified atmosphere of 8% CO2 in air. At 18 hours post-transfection, 1.5 mL of ExpiFectamin

CHO Transfection Enhancer

1 and 60 mL of ExpiFectamin CHO Transfection Feed were added to each flask. Each bispecific antibody was purified from the cell culture supernatant by recombinant Protein A affinity chromatography (Hitrap Mabselect Sure, GE Healthcare, 28-4082-55) and gel filtration chromatography with a HiLoad 26/200 Superdex200 prep grade column (GE Healthcare, 28-9893-36). SDS-PAGE (NuPage 4-12% Bis-Tris gel, NP0321) and size exclusion HPLC (Agilent, 1200 series) analysis with SE-HPLC column (SWXL SE-HPLC column, TOSOH, G3000SWXL) were performed to detect and confirm the size and purity of each bispecific antibody. Purified proteins were concentrated in PBS by ultrafiltration using a Amicon Ultra 15 30K device (Merck, UFC903096), and protein concentrations were estimated using a nanodrop (Thermo, Nanodrop One). When a two-vector system is applied, the ratio between light to heavy chain could be 1:1 to 1:3 by weight. Alternatively, a one-vector system that contains both chains in one single vector can also be used.

The prepared anti-ROR1/anti-4-1BB bispecific antibodies are named as A2F2M1x1A10M12, A2F2M1x1A12M1, BA6x1A10M12 and BA6x1A12M1, respectively, wherein the former refers to the clone in the IgG form and the latter refers to the clone in the scFv form.

Example 4. Characterization of bispecific antibodies ROR1x4-1BB

4.1. Binding of the bispecific antibodies to ROR1 and 4-1BB

4.1.1. SACE

To evaluate the binding activity of the bispecific antibodies prepared in Example 3 to either of ROR1 and 4-1BB, the bispecific antibodies were subjected to SACE (Single antigen capture ELISA) test. Briefly, microtiter plates were coated with human ROR1 or 4-1BB protein at 0.1μg/ml in PBS, 100 μl/well at 4°C overnight, then blocked with 100 μl/well of 1% BSA. Five-fold dilutions of antibodies were added to each well and incubated for 1-2 hours at RT. The plates were washed with PBS/Tween and then incubate with goat-anti-human IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 1 hour at RT. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630nm. As shown in FIGS. 13a and 13b, and Table 23, the bispecific antibodies tested show ROR1 and 4-1BB binding abilities.

EC50 (nM)	ROR1	4-1BB
A2F2M1(NA)x1A10 M12	0.00724	0.00772
A2F2M1(NA)x1A12 M1	0.0092	0.0111
BA6(NA)x1A10 M12	0.00841	0.085
BA6(NA)x1A12 M1	0.00983	0.0128

4.1.2. DACE

To evaluate the binding activity of the bispecific antibodies prepared in Example 3 to ROR1 and 4-1BB, the bispecific antibodies were subjected to DACE (Dual antigen capture ELISA) test. Briefly, microtiter plates were coated with 100 ng/well of human ROR1-Fc protein (Sinobio, 10084-H02H) in PBS at 4°C overnight, then blocked with 100 μl/well of 1% BSA for 2 hours at 37°C. Three-fold dilutions of each of the bispecific antibodies starting from 100 nM were added to each well and incubated for 2 hours at 37°C. The plates were washed with PBS/Tween and then incubate with 80 ng/well of human 4-1BB-His protein (Sinobio, 16498-H08H) in 1% BSA for 1 hour at 37°C. The plates were washed with PBS/Tween and then incubate with Anti-His HRP (Roche, Cat: 11965085001) for 1 hour at 37°C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-650nm. The results are shown in FIGS. 14a and 14b. As shown in FIGS. 14a and 14b, all the bispecific antibodies tested can simultaneously bind to both of human ROR1 and human 4-1BB proteins with high activities.

4.2. Binding of the bispecific antibodies to ROR1 on the surface of cancer cells

To evaluate the binding activity of the bispecific antibodies prepared in Example 3 to human ROR1 proteins expressed on the surfaces of various human cancer cells, the mean florescence intensity (MFI) was evaluated by FACSAriaIII. As shown in Table 24, the bispecific antibodies tested could bind to cell surface expressed human ROR1 proteins.

[Table 24]

4.3. Binding affinity of the bispecific antibodies

To evaluate the binding activity of the bispecific antibodies prepared in Example 3 to human ROR1 and 4-1BB proteins, this example performed the affinity ranking by using OCTET. As shown in Table 15 below, all the bispecific antibodies tested showed high 4-1BB binding affinity.

Antigen	Species (Antigen)	Sample	KD (M)	kon (1/Ms)	kdis (1/s)	chi	R²
ROR1	Human	A2F2M1(NA)x1A10 M12	1.27E-10	1.89E+06	2.40E-04	0.108	0.993
ROR1	Human	BA6(NA)x1A10M12	1.13E-10	1.12E+06	1.27E-04	0.204	0.992
4-1BB	Human	A2F2M1(NA)x1A10M12	7.18E-11	1.07E+06	7.71E-05	0.005	1.000
4-1BB	Human	BA6(NA)x1A10M12	9.23E-11	8.80E+05	8.12E-05	0.015	0.999

Example 5. 4- 1BB signal activation depending on ROR1 expression

In this assay, GloResponseTM NFκB-luc2/4-1BB Jurkat cell line, genetically modified to stably express human 4-1BB and luciferase downstream of a response element, was used as effector cell and cancer cells expressing or not expressing ROR1 were used as target cells. Urelumab (anti-4-1BB antibody) was used as a control group (BMUR). In brief, NCI-N87 (expressing ROR1) or BT474 (not expressing ROR1) were plated in a 96-well assay plate and cultured overnight. On the day of assay, the antibody to be tested and effector Jurkat cells were added to the plate. After 6 hrs incubation, Bio-GloTM Reagent was added and luminescence was measured using a microplate reader. The obtained results are shown in following FIGS. 15a (NCI-N87) and 15b (BT474).

As shown in FIGS. 15a and 15b, only bispecific antibodies showed 4-1BB signal activation when co-cultured with ROR1 high expressing cell. Cytokines were not secreted in cells with low ROR1 expression.

Example 6. T cell immune response

6.1. Effect on release of cytokine in over-expressing cell lines

Human PBMCs were co-cultured with MC38 cells which highly express ROR1 or not. Briefly, microplates were coated with 5 ug/mL of anti-CD3 (UCHT1) in PBS at 37°C for 2hr. Then human PBMC and MC38, MC38-human ROR1 expressed cells were plated in 96 well plate. Four-fold dilution of each of the antibodies starting from 20 nM were added to each well and incubated for 72hr. After 72hr incubation, the concentration of IFN-gamma in supernatant was measured by Human IFN-gamma Quantikine Kit (R&D system, SIF50). The obtained results are shown in following FIG. 16.

As shown in FIG. 16, bispecific antibodies induced cytokine release when co-cultured with ROR1 high expressing cell. The combined treatment of anti-4-1BB monoclonal antibodies and anti-ROR1 monoclonal antibodies did not induce cytokines.

6.2. Effect on target cell lysis and release of cytokine in cancer cells

NCI-N87 is gastric cancer cell lines expressing human ROR1. Human PBMCs were co-cultured with human ROR1 expressed cancer cells. Briefly, microplates were coated with 5ug/mL of anti-CD3(UCHT1) in PBS at 37°C for 2hr. Then human PBMC and NCI-N87 were plated in 96 well plate. Four-fold dilution of each of the antibodies starting from 20nM were added to each well and incubated for 72hr. After 72hr incubation, the concentration of IFN-gamma in supernatant was measured by Human IFN-gamma Quantikine Kit (R&D system, SIF50). And the survival of NCI-N87 was measured by cell counting kit-8 (Dojindo, CK04-20). The obtained results are shown in following FIG. 17 (E:T: Effector cell: Target cell ratio).

As shown in FIG. 17, the bispecific antibodies induced more cytokine release and target cell lysis than the combination of each monoclonal antibodies in presence of ROR1 high expressing cells.

Example 7. CD8 positive T cells response promoted by BsAb

7.1. Effect on release of cytokine

Purified isolated human CD8⁺ T cells (Stem cells, 70027) were co-cultured with NCI-N87 expressing ROR1. Human CD8⁺ T cells are effector cells and NCI-N87 is a target cell. It was used in experiments with various rates of effector cells and target cells. Briefly, microplates were coated with 5ug/mL of anti-CD3(UCHT1) in PBS at 37°C for 2hr. Then human CD8⁺ T cells and NCI-N87 were plated in 96 well plate. The antibodies were added to each well and incubated for 72hr. After 72hr incubation, the concentration of IFN-gamma and IL-2 in supernatant was measured by Human IFN-gamma Quantikine Kit (R&D system, SIF50) and Human IL-2 Quntikine kit (R&D system, S2058). The obtained results are shown in FIG. 18 (E:T: Effector cell: Target cell ratio).

As shown in FIG. 18, cytokines secretion was induced by CD8⁺ T cells. And bispecific antibodies induced more cytokine release than the combination of each monoclonal antibody in presence of ROR1 high expressing cells.

7.2. Effect on target cell lysis

Purified isolated human CD8⁺ T cells (Stem cells, 70027) were co-cultured with NCI-N87 expressing ROR1. Briefly, microplates were coated with 5ug/mL of anti-CD3(UCHT1) in PBS at 37°C for 2hr. Then human CD8+ T cells and NCI-N87 were plated in 96 well plate. The antibodies were added to each well and incubated for 72hr. After 72hr incubation, the survival of NCI-N87 was measured by cell counting kit-8 (Dojindo, CK04-20). The obtained results are shown in FIG. 19 (E:T: Effector cell: Target cell ratio).

As shown in FIG. 19, the bispecific antibodies induced more target cell lysis than the combination of each monoclonal antibody in presence of ROR1 high expressing cells.

Example 8. In vivo anti-tumor efficacy in 4-1BB knock-in mice

In vivo anti-tumor efficacy of anti-ROR1/anti-4-1BB bispecific antibodies was evaluated in human ROR1/MC38 tumor (Biocytogen) bearing h4-1BB knock-in mice (Biocytogen). Tumor bearing humanized mice were randomized to each test group(n=7~8/group) at day 8 Post tumor implantation based on tumor volume (approximately 80mm³). hIgG1 antibody (22.5mg/kg), and anti-ROR1/anti-4-1BB bispecific antibodies (A2F2M1(NA)x1A10 M12, BA6(NA)x1A10 M12, 10mg/kg and 30mg/kg, respectively) were respectively intra peritoneally administrated twice a week into the mice for 5 weeks. Tumor size was measured with a digital caliper.

The obtained results are shown in FIG. 20. As shown in FIG. 20, anti-ROR1/anti-4-1BB bispecific antibodies showed potent anti-tumor efficacy in human ROR1/MC38 tumor and some mice showed complete regression of the tumor.

The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

A anti-ROR1/anti-4-1BB bispecific antibody, comprising:

(a) an anti-ROR1 antibody or an antigen-binding fragment thereof comprising:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 105;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 106;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, or 23;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 107;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 108; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 109; and

(b) an anti-4-1BB antibody or an antigen-binding fragment thereof comprising:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 110;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 111;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 83 or 73;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 76;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 77; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 78.
The anti-ROR1/anti-4-1BB bispecific antibody of claim 1, comprising:

(a) an anti-ROR1 antibody or an antigen-binding fragment thereof comprising:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, or 23;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 31;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, or 39; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 40, 41, 42, 43, or 44; and

(b) an anti-4-1BB antibody or an antigen-binding fragment thereof comprising:

a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 68 or 69;

a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 70 or 71;

a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 72, 73, 74, or 75;

a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 76;

a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 77; and

a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 78.
The anti-ROR1/anti-4-1BB bispecific antibody of claim 1 or claim 2, wherein the anti-ROR1 antibody or an antigen-binding fragment thereof comprises:

a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54; and

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, or 63.
The anti-ROR1/anti-4-1BB bispecific antibody of claim 1 or claim 2, wherein the anti-4-1BB antibody or an antigen-binding fragment thereof comprises:

a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 79, 80, 81, 82, 84, 98, 99. 100, 101, or 102; and

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 85, 86, 103, or 104.
The anti-ROR1/anti-4-1BB bispecific antibody of claim 1 or claim 2, wherein the anti-4-1BB antibody or an antigen-binding fragment thereof is a scFv comprising:

a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 79, 80, 81, 82, 84, 98, 99. 100, 101, or 102;

a light chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 85, 86, 103, or 104; and

a peptide liker between the heavy chain variable region and the light chain variable region.
The anti-ROR1/anti-4-1BB bispecific antibody of claim 5, wherein the anti-ROR1 antibody is a full-length antibody.
A pharmaceutical composition comprising the anti-ROR1/anti-4-1BB bispecific antibody of claim 1 or claim 2.
The pharmaceutical composition of claim 7, which is for stimulating an immune response.
The pharmaceutical composition of claim 7, which is for treating or preventing a disease associated with ROR1, 4-1BB, or both thereof.
The pharmaceutical composition of claim 9, wherein the disease associated with ROR1, 4-1BB, or both thereof is a cancer, an infectious disease, an autoimmune reaction, or a nervous system disorder.
A method of stimulating an immune response or treating or preventing a disease associated with ROR1 in a subject in need thereof, comprising administering a pharmaceutically effective amount of the bispecific antibody of claim 1 or 2 to the subject.
A polynucleotide encoding the anti-ROR1/anti-4-1BB bispecific antibody of claim 1 or 2.
A recombinant vector or cell comprising the polynucleotide of claim 12.
A method of preparing the bispecific antibody, comprising expressing the polynucleotide of claim 12 in a cell.