RU2844869C1 - Anti-ror1 antibodies and related bispecific binding proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, in particular to antibodies recognizing orphan receptor 1 like receptor tyrosine kinase (ROR1), bispecific ROR1/CD3-binding proteins. Isolated antibody or its antigen-binding fragment specifically binds to ROR1 and contains CDR-H1 with SEQ ID NO: 1, CDR-H2 with SEQ ID NO: 2 or SEQ ID NO: 4, CDR-H3 with SEQ ID NO: 3, CDR-L1 with SEQ ID NO: 5, CDR-L2 with SEQ ID NO: 6 and CDR-L3 with SEQ ID NO: 7. Also disclosed are a conjugate, a nucleic acid molecule and a host cell for producing a bispecific binding protein, a pharmaceutical composition for treating a disorder, at which ROR1 activity is malicious. Antibody is applicable for detecting human ROR1 or human CD3, for inhibiting the ROR1 signalling pathway and/or for inhibiting human ROR1-mediated tumour growth or metastasis.

EFFECT: bispecific polyvalent binding proteins are applicable for inducing ROR1-specific T-cell cytotoxicity and/or anticancer activity against ROR1-expressing malignant cells.

24 cl, 11 dwg, 16 tbl, 11 ex

Description

Field of technology

The present invention relates to antibodies capable of recognizing the orphan receptor 1, receptor tyrosine kinase-like (ROR1) and related bispecific binding proteins, for example, ROR1/CD3 bispecific binding proteins (e.g., FIT-Ig and MAT-Fab binding proteins). The antibodies and bispecific binding proteins described in the present application can be used to treat diseases such as hematopoietic cancers and solid tumors.

State of the art

Orphan receptor tyrosine kinase-like 1 (ROR1) is an evolutionarily conserved type I membrane protein belonging to the ROR subfamily. Its amino acid (AA) sequence is 58% identical to that of ROR2, the second and final member of the ROR family. ROR1 and ROR2 consist of a distinct extracellular region with one immunoglobulin-like (Ig-like) domain, one frizzled (Fz), and one kringle (Kr) domain, followed by a transmembrane region and an intracellular region containing the tyrosine kinase domain (Baskar, S., et al., (2008) Clinical Cancer Research, 14(2), 396-404).

ROR1 expression is developmentally regulated and attenuated during fetal development. Gene expression profiling of B-cell malignancies and normal B lymphocytes led to the discovery of ROR1 and its characteristic expression in lymphocytic leukemia cells (see Baskar et al., 2008, supra). Using the highly sensitive mouse anti-human ROR1 mAb 6D4, ROR1 was characterized as a typical membrane protein uniformly expressed in certain solid tumor types, including ovarian cancer, triple-negative breast cancer, lung adenocarcinoma, and pancreatic adenocarcinoma. Furthermore, cell surface expression of ROR1 was observed in some normal tissues (e.g., parathyroid gland, pancreatic islets, and several regions of the human intestine), but not in other tissues (e.g., brain, heart, lung, and liver) (Berger et al., 2016, Clinical Cancer Research, 23(12), 3061-3071).

ROR1 has been proposed as a target for cancer treatment. For example, WO2005100605, WO2007051077, WO2008103849 and WO2012097313 describe anti-ROR1 antibodies and their use as therapeutic agents for targeting tumors, including solid tumors such as breast cancer and hematological tumors such as chronic lymphocytic leukemia (CLL). Cirmtuzumab, obtained by mapping the epitope bound by the anti-ROR1 antibody D10 of WO2012097313, is a humanized monoclonal antibody used in clinical trials of various types of cancers, including chronic lymphocytic leukemia (CLL). Cirmtuzumab blocks the binding of ROR1 to its ligand Wnt5a, which can inhibit Wnt5a-induced stimulation of NF-κB activation and thereby suppress autocrine IL-6-dependent STAT3 activation in CLL (Chen et al., 2019, Blood, 134(13), 1084-1094). Cirmtuzumab can be internalized into cells and has been evaluated for use as a targeting moiety in anti-ROR1 antibody drug conjugates (ADCs). A cirmtuzumab-based ADC VLS-101 containing MMAE was developed for the treatment of patients with ROR1-positive malignancies.

Another treatment option has been the development of bispecific antibodies against ROR1 and a second antigen, such as bispecific T cell engager activators (BiTEs). WO 2014/167022 describes a bispecific antibody with the slowly internalizing ROR1 antibody R12 as one arm and an anti-CD3e antibody as the other arm. Gohil et al., 2017 (Onco Immunology, 6(7), 1-11) used single-chain variable fragments (scFv) targeting the frizzled domain of ROR1 to generate BiTEs that prevented pancreatic tumor xenograft engraftment in mouse models. In the article by Qi et al., 2018 (Proceedings of the National Academy of Sciences of the United States of America, 115(24), E5467-E5476), an scFv with a membrane-proximal epitope R11 targeting ROR1 was described, which showed potent and selective antitumor activity when constructed in the scFv-Fc format using a bispecific ROR1×CD3 antibody based on a heterodimeric and aglycosylated Fc domain.

BiTEs are bispecific antibodies against the constant component of the T cell/CD3 complex and tumor-associated antigen (TAA). These bispecific antibodies have certain advantages, such as the ability to redirect the cytotoxic activity of T cells to malignant cells without MHC restriction. The clinical success of blinatumomab in recent years has generated increasing interest in the use of CD3-targeted BiTEs for cancer immunotherapy. However, concerns have arisen regarding the efficacy and toxicity/safety of this treatment approach.

For example, it may be desirable to have an antibody with increased affinity for antigens that are strictly tumor-specific. However, in the case of a tumor-associated antigen that is overexpressed in tumors as well as expressed in normal tissues, the ability of the antibody to discriminate between tumor and normal tissue expression of the antigen may be advantageous. The internalization properties of an antibody may also influence its therapeutic use. Strong internalization upon antibody binding, for example, may be desirable for antibody conjugates to efficiently deliver the conjugated toxin to target cells. However, internalization may be a disadvantageous property for T-cell-attracting activators, since retention of BiTE on the cell surface may be desirable to provide cytotoxic activity by engaging T cells. In addition, it has been shown that the affinity and ability of the antibody to induce antigen internalization may influence the penetration into solid tumors and the efficacy of antibody-based drugs. According to Rudnick et al., 2011 (Rudnick et al., Cancer Res; 71(6); 2250-9), high affinity and rapid internalization may limit the penetration of the antibody into the tumor, while relatively low affinity and reduced internalization capacity may lead to more efficient penetration into solid tumors.

The art mentions many factors that influence the in vivo activity and tumor selectivity of BiTE. Different attributes may often be desirable for a T cell attractant depending on the nature of the target/epitope.

The paper by James et al. (Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane, J. Immunol. 180 (10) (2008) 7028–7038), for example, reported that modulation of the epitope distance from the membrane improves the tumor potency and/or selectivity of BiTE. By examining CD22 epitope targeting at different membrane distances with CAR-T cells, James et al found that targeting the intermediate domain resulted in efficient lysis of target B cell lines, while no lysis of normal B cells was detected. Similarly, Qi et al. found that epitope location on ROR1 can influence the activity of ROR1xCD3 bispecific antibodies in scFv-Fc format (see Qi et al., 2018, supra). Data from screening a panel of mAbs with different epitopes against ROR1 by Qi et al suggest that the membrane-proximal epitope in the Kr domain of ROR1, targeted by R11, may be a suitable site for T-cell recruitment by bispecific antibodies, whereas the membrane-distal epitope at the junction of the Fz and Kr domains, targeted by R12, may not be such a site. A bispecific antibody with the R12 arm demonstrated only weak antitumor activity in vivo.

Various approaches to enhance tumor cell preferential engagement by engineering the antibody format have been described, including those based on its size, valence, and geometry. Slaga et al. (Avidity-based binding to HER2 results inselective killing of HER2-overexpressing cells by anti-HER2/CD3, Sci. Transl. Med. 10 (463) (2018)) explored an avidity-based strategy using a multivalent antibody format and designed a bispecific antibody with affinities selected to enhance the discrimination of cells expressing HER2 at low or high density. G.L. Moore, et al. (A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats, Methods (2018)) reported a similar strategy.

In addition, an issue to consider when developing a bispecific antibody is its suitability for manufacture. Low production yield and a strong tendency to form aggregates are properties that may make an antibody-based drug impractical for preclinical and clinical evaluation stages.

In light of the above, and given that ROR1 is a promising target for cancer treatment, there remains a need in the art to develop diverse anti-ROR1 molecules with different binding efficiencies and/or binding sites or internalization properties, to develop diverse antibody formats, and to broaden and/or improve therapeutic utility and manufacturability.

Brief description of the invention

The present invention addresses the above needs by providing novel anti-ROR1 antibodies, anti-CD3 antibodies, and engineered bispecific proteins that bind both ROR1 and CD3.

In particular, in some embodiments, the present invention relates to antibodies against ROR1, e.g., antibodies with high binding affinity to cells expressing ROR1 and a low internalization rate. In some embodiments, the present invention also provides antibodies that bind to CD3, e.g., antibodies that bind to CD3 with high affinity. In some embodiments, the present invention also provides a bispecific ROR1/CD3-CBfl3biBaron5ift protein in a tandem Fab immunoglobulin (FIT-Ig) format or in a monovalent asymmetric tandem Fab bispecific antibody (MAT-Fab) format that is reactive with both ROR1 and CD3. In some embodiments, the antibodies of the present invention are useful for detecting human ROR1 or human CD3, inhibiting the ROR1 signaling pathway, and/or suppressing human ROR1-mediated tumor growth or metastasis, in vitro or in vivo. Furthermore, in some embodiments, the bispecific multivalent binding proteins described herein are useful for inducing ROR1-specific T cell cytotoxicity and/or potent antitumor activity against ROR1-expressing malignant cells in vivo.

In some embodiments, the present invention also provides methods for making and using the anti-ROR1 antibodies and anti-CD3 antibodies and bispecific ROR1/CD3 binding proteins described herein. In addition, various compositions are described, such as compositions that can be used in methods for detecting ROR1 and/or CD3 in a sample or in methods for treating or preventing a disorder in an individual associated with ROR1 and/or CD3 activity.

Brief description of the drawings

Figure 1 shows the binding activity of ROR1-ECD protein by monoclonal antibodies. Non-specific mIgG1 was used as a negative control.

Figures 2A-B illustrate the binding activity of anti-ROR1 monoclonal antibodies to ROR1-expressing cells. Non-specific mIgG1 was used as a negative control.

Figure 3 shows the CD3 binding efficiency of the ROR1×CD3 bispecific antibodies compared to the corresponding parental anti-CD3 monoclonal antibodies. Non-specific hIgG was used as a negative control.

Figures 4A-D illustrate the binding efficiency of ROR1 by the ROR1×CD3 bispecific proteins and their common parental anti-ROR1 monoclonal IgG1 antibody (HuROR1-mAb004-1) against ROR1-expressing tumor cells (A) NCI-H1975, (B) MDA-MB-231, (C) A549, and (D) RPMI-8226.

Figure 5 shows the results of a co-culture reporter gene assay measuring redirected CD3 activation by ROR1×CD3 bispecific FIT-Ig and MAT-Fab antibodies compared to monospecific anti-CD3 IgG (HuEM1006-01-24 and HuEM1006-01-27) and non-specific FIT-Ig (EMB01).

Figure 6 shows the results of a Jurkat-NFAT-luc-based reporter gene assay testing off-target redirected CD3 activation by humanized ROR1×CD3 bispecific antibodies compared to monospecific anti-CD3 IgG (HuEM1006-01-24 and HuEM1006-01-27) and non-specific FIT-Ig (EMB01).

Figure 7 shows the results of the T cell redirected cytotoxicity assay using different ROR1×CD3 bispecific antibodies. Non-specific FIT-Ig (EMB01) was used as a negative control.

Figure 8 shows the tumor volume profile of MDA-MB-231 in M-NSG mice transplanted with human PBMCs treated with ROR1×CD3 bispecific antibodies or vehicle control.

Figures 9A-C show the results of the internalization assay using the humanized anti-ROR1 antibody and the bispecific antibodies (A) HuROR-mAb004-1, (B) FIT 1007-12B-17, and (C) MAT1007-12B-17.

Figure 10A shows a schematic illustration of the domain structure of the bispecific antibody FIT-Ig in LH format and HL format. Figure 10B shows a schematic illustration of the domain structure of the bispecific antibody MAT-Fab in LH format and HL format.

Figure 11A shows the binding activity of FIT-Ig molecules to ROR1-expressing MDA-MB-231 cells. Figure 11B shows the binding activity of FIT-Ig molecules to CD3-expressing Jurkat cells. Figure 11C shows the results of a T cell redirected cytotoxicity assay comparing FIT1007-12B-17 to two reference FIT-Ig molecules.

Detailed description of the invention

The present invention relates to anti-ROR1 antibodies, anti-CD3 antibodies, antigen-binding fragments thereof, and multivalent bispecific binding proteins, e.g., FIT-Ig or MAT-Fab, that bind to both ROR1 and CD3. Various aspects of the present invention relate to anti-ROR1 and anti-CD3 antibodies and antibody fragments, FIT-Ig and MAT-Fab binding proteins that bind to human ROR1 and human CD3, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such antibodies, functional antibody fragments, and binding proteins. The present invention also encompasses methods of using the antibodies, functional antibody fragments, and bispecific binding proteins of the present invention to detect human ROR1 and/or human CD3; modulate the activity of human ROR1 and/or human CD3 in vitro or in vivo; and treatment of diseases, especially cancer, mediated by the binding of ROR1 and CD3 to their respective ligands.

Definitions

Unless otherwise defined in this application, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. In case of hidden ambiguity, the definitions provided in this application take precedence over any dictionary or external definition. Furthermore, unless the context otherwise requires, singular references to terms include the plural, and plural references include the singular. In this application, the use of "or" means "and/or" unless otherwise specified. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Furthermore, terms such as "element" or "component" include both elements and components comprising a single block and elements and components comprising more than one block, unless otherwise explicitly specified.

In the present application, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered in accordance with the Kabat numbering system, which is described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to in this application as the "Kabat numbering." In particular, the Kabat numbering system (see pp. 647-660) Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of the kappa and lambda isotypes, and the Kabat-EC index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which in this case is further clarified by reference to "Kabat-EC index numbering").

General information on the sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source, is not associated with the natural components that accompany it in its native state, is substantially free of other proteins of an organism of the same species, is expressed by a cell of an organism of another species, or is not found in nature. A polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates may be "isolated" from the components associated with it in natural conditions. A protein may also be substantially free of the components associated with it in natural conditions by isolation using protein purification techniques well known in the art.

The term "specific binding" or "specifically binding" when used with respect to the interaction of an antibody, binding protein, or peptide with a second chemical entity means that said interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical entity. For example, an antibody recognizes and binds to a particular protein structure rather than to proteins in general. In general, if an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction mixture containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "antibody" broadly refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof that retains the essential epitope-binding properties of the Ig molecule. Such mutant, variant or derivative antibody formats are known in the art, and non-limiting embodiments are discussed below.

In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second, and third CDRs of the VH domain are typically numbered CDR-H1, CDR-H2, and CDR-H3; similarly, the first, second, and third CDRs of the VL domain are typically numbered CDR-L1, CDR-L2, and CDR-L3. Immunoglobulin molecules can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

The term "Fc region" is used to refer to the C-terminal region of an immunoglobulin heavy chain that can be obtained by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin typically contains two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally contains a CH4 domain, such as in the case of the Fc regions of IgM and IgE antibodies. The Fc region of IgG, IgA, and IgD antibodies contains a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies does not contain a hinge region, but contains a CH2 domain, a CH3 domain, and a CH4 domain. Variant Fc regions comprising substitutions of amino acid residues in the Fc region to alter the effector function of an antibody are known in the art (see, e.g., Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). The Fc region of an antibody may mediate one or more effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and/or the half-life of the antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for a therapeutic antibody, but in other cases, depending on the therapeutic goals, may be unnecessary or even detrimental. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγR and the complement protein Clq, respectively. In another embodiment, at least one amino acid residue in a constant region of an antibody, such as an Fc region of an antibody, is substituted in a manner that affects the effector functions of the antibody. Dimerization of two identical immunoglobulin heavy chains is mediated by dimerization of the CH3 domains and is stabilized by disulfide bonds in the hinge region that connects the CH1 constant domains to the Fc constant domains (e.g., CH2 and CH3). The anti-inflammatory activity of IgG is dependent on sialylation of the N-linked glycan of the Fc portion of IgG. The precise glycan requirements for anti-inflammatory activity have been defined, so that a suitable IgG1 Fc fragment can be engineered, thereby producing a fully recombinant sialylated IgG1 Fc with significantly enhanced activity (see Anthony et al., Science, 320: 373–376 (2008)).

The terms "antigen-binding portion" and "antigen-binding fragment" or "functional fragment" of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, i.e., the same antigen (e.g., ROR1, CD3), as the full-length antibody from which the fragment is derived. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Such embodiments of the antibody can also be bispecific, dual-specific, or multispecific formats that specifically bind to two or more different antigens (e.g., ROR1 and another antigen, e.g., CD3). Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the Fd fragment, consisting of the VH and CH1 domains; (iv) the Fv fragment, consisting of the VL and VH domains of one arm of an antibody, (v) the dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT Publication No. WO 90/05144) containing one variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by recombinant techniques through a synthetic linker, allowing them to be produced as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Set USA, 85: 5879-5883 (1988)). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody and the equivalent terms given above. This term also encompasses other forms of single-chain antibodies, such as diabodies. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains on one chain, resulting in the promotion of pairing of the domains with the complementary domains of the other chain and the creation of two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444–6448 (1993). Such antibody binding fragments are known in the art (Kontermann and Dtibel eds., Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5)). In addition, single-chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and U.S. Patent No. 5,641,870)).

The constant domain of an immunoglobulin (C) refers to the constant domain of a heavy (CH) or light (CL) chain. The amino acid sequences of the constant domain of the heavy chain and light chain of mouse and human IgG are known in the art.

The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, targeting a single antigenic determinant (epitope). Furthermore, unlike polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each mAb targets a single antigenic determinant. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method.

The term "human sequence" in relation to the constant domain of the light chain CL, the constant domain of the heavy chain CH and the Fc region of an antibody or binding protein according to the present application means that said sequence belongs to or is derived from a human immunoglobulin sequence. The human sequence according to the present invention can be a native human sequence or a variant thereof comprising an alteration of one or more (e.g. up to 20, 15, 10) amino acid residues.

The term "chimeric antibody" refers to antibodies that contain heavy and light chain variable region sequences from one animal species and constant region sequences from another animal species, such as antibodies containing mouse heavy and light chain variable regions linked to human constant regions.

The term "CDR-grafted antibody" refers to antibodies comprising heavy and light chain variable region sequences from one animal species in which sequences from one or more of the VH and/or VL CDR regions have been replaced by CDR sequences from another animal species, such as antibodies comprising human heavy and light chain variable regions in which one or more of the human CDR regions have been replaced by mouse CDR sequences.

The term "humanized antibody" refers to antibodies comprising heavy and light chain variable region sequences from a non-human animal (e.g., a mouse) in which at least a portion of the VH and/or VL sequence has been altered to increase its "human-likeness," i.e., similarity to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which non-human (e.g., a mouse) CDR sequences are incorporated into human VH and VL framework sequences. A humanized antibody is an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds an antigen of interest and comprises framework regions and constant regions that comprise substantially the amino acid sequence of a human antibody, with complementarity determining regions (CDRs) comprising substantially the amino acid sequence of an antibody of non-human origin. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of a CDR of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') ₂ , Fv), in which all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin (i.e., a donor antibody), and all or substantially all of the framework regions are regions of a human immunoglobulin consensus sequence. In one embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. In some embodiments, the humanized antibody comprises both a light chain and at least a heavy chain variable domain. The antibody may also comprise CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In particular embodiments, the humanized antibody comprises only a humanized light chain variable domain and/or a humanized heavy chain.

The humanized antibody can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any isotype, including, but not limited to, IgG1, IgG2, IgG3 and IgG4. The humanized antibody can contain sequences of more than one class or isotype, and specific constant domains can be selected to optimize the desired effector functions using techniques well known in the art.

The framework regions and CDR regions of a humanized antibody need not correspond exactly to the original sequences, for example, a CDR of a donor antibody or an acceptor framework can be mutated by substitution, insertion and/or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In a typical embodiment, such mutations are not extensive, however. Typically, at least 80%, at least 85%, at least 90% or at least 95% of the residues of the humanized antibody correspond to residues of the original FR and CDR sequences. In order to maintain a particular loop structure or to properly orient the CDR sequences to contact the target antigen, back mutation at a particular position of the framework region is often used to restore the same amino acid that is at that position in the donor antibody.

The term "CDR" refers to the complementarity determining regions in the variable domain sequences of an antibody. Three CDRs are present in each of the variable regions of the heavy chain and light chain, designated CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. As used herein, the term "set of CDRs" refers to a group of three CDRs occurring in a single variable region capable of binding antigen. The precise boundaries of these CDRs are defined differently depending on the different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) provides not only an unambiguous residue numbering system applicable to any antibody variable region, but also the precise boundary residues defining the three CDRs.

The term "Kabat numbering" with respect to the CDRs of an antibody heavy and light chain, as is well recognized in the art, refers to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chain of an antibody or antigen-binding fragment thereof. See Kabat et al., Ann. NY Acad. Set, 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

The growth and analysis of large publicly available databases of amino acid sequences of heavy and light chain variable regions over the past twenty years has led to an understanding of the typical boundaries between framework regions (FRs) and CDR sequences within variable region sequences and has allowed those skilled in the art to accurately assign CDRs according to Kabat numbering, Chothia numbering, or other systems. See, e.g., Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," In Kontermann and Dübel, eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pages 432-433.

The term "multivalent binding protein" refers to a binding protein that contains two or more antigen-binding sites. A multivalent binding protein is in some cases engineered to contain three or more antigen-binding sites and is typically not a naturally occurring antibody. The term "bispecific binding protein" (which may be used interchangeably with the term "bispecific antibody" unless otherwise specified) refers to a binding protein that is capable of binding two targets with different specificities. The FIT-Ig binding proteins of the present invention contain four antigen-binding sites and are typically tetravalent binding proteins. The MAT-Fab binding proteins of the present invention contain two antigen-binding sites and are typically bivalent binding proteins. The FIT-Ig or MAT-Fab of the present invention binds both ROR1 and CD3 and is bispecific.

The FIT-Ig binding protein, comprising two long (heavy) V-C-V-C-Fc- polypeptide chains and four short (light) V-C polypeptide chains, forms a hexamer displaying four Fab antigen-binding sites (VH-CH1 paired with VL-CL, sometimes referred to as VH-CH1::VL-CL). Each half of the FIT-Ig comprises a heavy chain polypeptide and two light chain polypeptides, and complementary pairing of the VH-CH1 and VL-CL immunoglobulin elements of the three chains results in the formation of two Fab-structured antigen-binding sites arranged in tandem. In the present disclosure, the immunoglobulin domains comprising the Fab elements are preferably directly fused into the heavy chain polypeptide without the use of interdomain linkers. That is, The N-terminal V-C element of long (heavy) polypeptide chains is directly fused at its C-terminus to the N-terminus of another V-C element, which in turn is linked to the C-terminal Fc region. In bispecific FIT-Ig binding proteins, tandem Fab elements are capable of reacting with different antigens. Each Fab antigen-binding site contains a heavy-chain variable domain and a light-chain variable domain, for a total of six CDRs per antigen-binding site.

A description of the design, expression and characterization of FIT-Ig molecules is provided in PCT publication WO 2015/103072. An example of such FIT-Ig molecules comprises a heavy chain and two different light chains. The heavy chain has the structural formula VL _A -CL-VH _B -CH1-Fc, where CL is directly fused to VH _B (namely, the "LH format") or VH _A -CH1-VL B -CL-Fc, where CH1 is directly fused to VL _B (namely, the "HL format"), and the two light polypeptide chains of the FIT-Ig are respectively characterized by the formulas VH _A -CH1 and VL _B -CL (for the "LH format") or VL _A -CL and VH _B -CH1 (for the "HL format"), respectively; wherein VL _A is a light chain variable domain of the parent antibody that binds antigen A, VL _B is a light chain variable domain of the parent antibody that binds antigen B, VH _A is a heavy chain variable domain of the parent antibody that binds antigen A, VH _B is a heavy chain variable domain of the parent antibody that binds antigen B, CL is a light chain constant domain, CH1 is a heavy chain constant domain, and Fc is an Fc region of an immunoglobulin (e.g., the C-terminal hinge-CH2-CH3 fragment of the heavy chain of an IgG1 antibody). In bispecific embodiments of FIT-Ig, antigen A and antigen B are different antigens or different epitopes of the same antigen. As used herein, one of A and B is ROR1 and the other is CD3, such as A is ROR1 and B is CD3.

The MAT-Fab binding protein, which contains one long (heavy) V-C-V-C-Fc polypeptide chain, two short (light) V-C polypeptide chains, and one immunologic Fc polypeptide chain, forms a tetramer displaying two Fab antigen-binding sites arranged in tandem (VH-CH1 paired with VL-CL, sometimes referred to as VH-CH1::VL-CL) and one Fc:Fc dimer. Modifications are often introduced into the CH3 domain of the Fc region of the MAT-Fab heavy chain (abbreviated CH3m1 domain) as well as the CH3 domain of the MAT-Fab Fc polypeptide chain (abbreviated CH3m2 domain) to favor heterodimerization of the two CH3 domains. The modifications may be knob-into-hole (KIH) mutations, for example, a mutation is made to create a structural knob in the CH3m1 domain of the heavy chain to pair with the CH3m2 domain of the Fc chain, which contains a complementary structural hole. However, other modifications, for example, modifications introduced into the domains by salt bridges or electrostatic interactions, are also useful. The constant regions may also contain other modifications, for example, Cys residues to stabilize the MAT-Fab molecule and/or mutations to prevent or reduce Fc effector functions. A feature of the structure of the bispecific MAT-Fab antibody described in the present application is preferably that all adjacent variable and constant domains of the heavy and light chains of the immunoglobulin are directly linked to each other without the participation of an intermediate synthetic amino acid or peptide linker.

A description of the design, expression and characterization of MAT-Fab molecules is provided in PCT Publication WO 2018/035084. One example of such MAT-Fab molecules comprises a heavy chain with a knob in the Fc region, two different light chains and one Fc polypeptide chain with a hole. In some embodiments, the heavy chain has the structural formula VL _A -CL-VH _B -CH1-hinge-CH2-CH3m1, where CL is directly fused to VH _B (namely, the "LH format"), or VH _A -CH1-VL _B -CL-Fc, where CH1 is directly fused to VL _B (namely, the "HL format"), and the two MAT-Fab light polypeptide chains are respectively characterized by the formulas VH _A -CH1 and VL _B -CL (for the "LH format") or VL _A -CL and VH _B -CH1 (for the "HL format"), respectively; wherein VL _A is a light chain variable domain of the parent antibody that binds antigen A, VL _B is a light chain variable domain of the parent antibody that binds antigen B, VH _A is a heavy chain variable domain of the parent antibody that binds antigen A, VH _B is a heavy chain variable domain of the parent antibody that binds antigen B, CL is a light chain constant domain, CH1 is heavy chain constant domain 1, and CH3m1 is heavy chain constant domain 3 with knob mutations such as S354C and T366W. The Fc polypeptide chain may be the C-terminal hinge-CH2-CH3 fragment of an immunoglobulin heavy chain (e.g., an IgG antibody) with hole mutations complementary to the knob mutations in CH3m2, such as T366S, L368A, and Y407V. In bispecific embodiments of the MAT-Fab, antigen A and antigen B are different antigens or different epitopes of the same antigen. As used herein, one of antigens A and B is ROR1 and the other is CD3, such as A is ROR1 and B is CD3.

In the present application, the term "k _on " (also "Kon", "kon") is intended to mean the rate constant of association of a binding protein (e.g., an antibody) with an antigen to form an associated complex, e.g., an antibody/antigen complex, as known in the art. The term "k _on " is also known by the terms "association rate constant" or "ka", which are used interchangeably in the present application. This value means the rate of binding of an antibody to a target antigen or the rate of formation of a complex between an antibody and an antigen, as shown in the equation below:

Antibody (“At”) + Antigen (“Ag”) → At-Ag.

In the present application, the term "k _off " (also "K _off ", "k _off ") is intended to mean the rate constant for dissociation or "dissociation rate constant" of a binding protein (e.g., an antibody) from an associated complex (e.g., an antibody/antigen complex), as known in the art. This value represents the rate of dissociation of an antibody from its target antigen or the partition of an Ab-Ag complex over time into free antibody and antigen, as shown in the equation below:

At+Ag ← At-Ag.

In this application, the term "K _D " (also "Kd") is intended to mean "equilibrium dissociation constant" and refers to the value obtained by measuring a titration at steady state or by dividing the dissociation rate constant (k _off ) by the association rate constant (k _on ). The association rate constant (k _on ), the dissociation rate constant (k _off ) and the equilibrium dissociation constant (K _D ) are used to represent the binding affinity of an antibody to an antigen. Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence techniques provides high sensitivity and the ability to study samples in physiological buffers at steady state. Other experimental approaches and instruments can be used, such as the BIAcore® (Biomolecular Interaction Assay) assay (e.g., an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Biolayer interferometry (BLI), using for example the Octet® RED96 system (Pall FortéBio LLC), is another method for affinity analysis. Additionally, the KinExA® (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, ID, USA) can be used.

The term "isolated nucleic acid" means a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that is, as a result of human intervention, not associated with all or part of the polynucleotides with which it occurs in nature; is operably linked to a polynucleotide with which it is not naturally associated; or is not naturally occurring as part of a larger sequence.

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell and are thus replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors suitable for use in recombinant DNA technology are often in the form of plasmids. In the present specification, the terms "plasmid" and "vector" may be used interchangeably, since the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors (e.g., retroviruses, adenoviruses, and replication-defective adeno-associated viruses), which serve equivalent functions.

The term "operably linked" refers to immediate proximity where the components described interact to allow them to function in the intended manner. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include both expression control sequences that are immediately adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences necessary to influence the expression and processing of the coding sequences to which they are ligated. Expression control sequences include appropriate transcriptional initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; cytoplasmic mRNA stabilizing sequences; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies with the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and a transcription termination sequence. The term "control sequences" is intended to include components whose presence is required for expression and processing, and may also include additional components whose presence is preferred, such as leader sequences and fusion partner sequences.

In the present application, "transformation" refers to any process by which exogenous DNA is introduced into a host cell. Transformation may occur naturally or artificially using various methods well known in the art. Transformation may be based on any known method of introducing foreign nucleotide sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the introduced DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome. They also include cells that transiently express the introduced DNA or RNA for a limited time.

The term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., several) nucleic acids encoding antibodies, such as the host cells described, for example, in U.S. Patent No. 7,262,028. Such terms are intended to refer not only to a particular cell of a subject, but also to the progeny of such a cell. Because certain modifications are possible in subsequent generations due to mutations or environmental influences, such progeny may not be substantially identical to the parent cell, but are still within the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from any kingdom of living organisms. In another embodiment, eukaryotic cells include protozoan, fungal, plant, and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; the mammalian cell lines CHO, HEK 293, COS, NS0, SP2, and PER.C6; the insect cell line Sf9; and the fungal cell line Saccharomyces cerevisiae.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent sufficient to reduce or alleviate the severity and/or duration of a disorder or one or more of its symptoms; prevent progression of the disorder; cause regression of the disorder; prevent recurrence, development, or progression of one or more of the symptoms associated with the disorder; cure the disorder; or enhance or improve the prophylactic or therapeutic effect(s) of another therapeutic agent (e.g., a prophylactic or therapeutic agent).

The antibodies, functional fragments thereof, and binding proteins of the present invention can be purified (for their intended use) using one or more of various methods and materials available in the art for purifying antibodies and binding proteins. Such methods and materials include, but are not limited to, affinity chromatography (e.g., using resins, particles, or membranes conjugated to Protein A, Protein G, Protein L, or a specific antibody ligand, functional fragment thereof, or binding protein), ion exchange chromatography (e.g., using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; e.g., using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (to inactivate contaminating viruses), and combinations thereof to obtain acceptable purity for the intended use. A non-limiting example of low pH treatment to inactivate contaminating viruses includes lowering the pH of a solution or suspension containing an antibody, functional fragment thereof, or binding protein of the present invention to pH 3.5 with 0.5 M phosphoric acid at 18°C to 25°C for 60-70 minutes.

Standard techniques (e.g., electroporation, lipofection) can be used to handle recombinant DNA, synthesize oligonucleotides, and culture and transform tissues. Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications or according to procedures conventional in the art or as described herein. The above techniques and procedures can generally be performed according to conventional methods well known in the art and described in various general or more specific references that are cited and discussed throughout the text of the present specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Monospecific antibodies against ROR1 and against CD3

The anti-ROR1 and anti-CD3 antibodies of the present invention can be produced by any of a number of methods known in the art. For example, by expression in host cells, wherein expression vector(s) encoding the heavy and light chains are transfected into the host cell by standard methods. The various forms of the term "transfection" are intended to cover a wide range of techniques commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, such as mammalian host cells, is specifically contemplated by the present invention since such eukaryotic cells (and in particular mammalian cells) assemble and secrete an immunologically active antibody with a proper three-dimensional structure.

In some embodiments, mammalian host cells for expressing the recombinant antibodies of the present invention are Chinese hamster ovary cells (CHO cells) (including ^dhfr cells as described in Urlaub and Chasin, Proc. Natl. Acad. Set USA, 11: 4216-4220 (1980), used with the DHFR selection marker, such as described in Kaufman and Sharp, J. Mol. Biol, 159: 601-621 (1982)), NSO myeloma cells, COS cells, and SP2 cells. When introducing recombinant expression vectors encoding antibody genes into mammalian host cells, the antibodies are produced by culturing the host cells for a time sufficient to allow expression of the antibody in the host cells or further secreting the antibody into the culture medium in which the host cells are grown. Antibodies can be isolated from the culture medium using standard protein purification methods.

Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be appreciated that variations of the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of a light chain and/or a heavy chain of an antibody of the present invention. Recombinant DNA technology can also be used to completely or partially remove DNA encoding one or both of the light and heavy chains that is not necessary for binding to antigens of interest. Molecules expressed from such truncated DNA molecules are within the scope of the term "antibody" of the present invention. Furthermore, bifunctional antibodies can be produced in which one heavy and one light chain are an antibody of the present invention and the other heavy and light chains are specific for an antigen other than the antigens of interest by cross-linking an antibody of the present invention with a second antibody using standard cross-linking chemistry.

In an exemplary system for recombinant expression of an antibody or antigen-binding fragment thereof according to the present invention, a recombinant expression vector encoding both an antibody heavy chain and an antibody light chain is introduced into dhfr ^- CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to CMV enhancer/AdMLP promoter regulatory elements to promote high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells transfected with the vector using methotrexate/amplification selection. The selected transfected host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare a recombinant expression vector, transfect host cells, select transfectants, culture host cells, and recover the antibody from the culture medium. In addition, the present invention provides a method for producing a recombinant anti-ROR1 antibody or an anti-CD3 antibody by culturing a transfected host cell according to the present invention in a suitable culture medium to obtain a recombinant antibody according to the present invention. The method may further comprise recovering the recombinant antibody from the culture medium.

Antibodies against ROR1

In some embodiments, the present invention provides antibodies that bind to ROR1 at the C-terminus of the Ig-like domain of ROR1. The antibodies described herein in some embodiments have high cell binding efficiency and/or are characterized by a low internalization rate, for example, as measured by a cell-based assay.

In some embodiments of the present invention, an isolated anti-ROR1 antibody or antigen-binding fragment thereof that specifically binds to ROR1 is described. In a further embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein:

- CDR-H1 contains the sequence RSWMN (SEQ ID NO: 1);

- CDR-H2 contains the sequence RIYPGNGDIKYNGNFKG (SEQ ID NO: 2) or RIYPGNADIKYNANFKG (SEQ ID NO: 4);

- CDR-H3 contains the sequence IYYDFYYALDY (SEQ ID NO: 3);

- CDR-L1 contains the sequence KASQDINKYIT (SEQ ID NO: 5);

- CDR-L2 contains the sequence YTSTLQP(SEQ ID NO: 6);

- CDR-L3 contains the sequence LQYDSLLWT (SEQ ID NO: 7),

wherein the said CDRs are defined in accordance with the Kabat numbering.

In some embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof comprises at positions H31-H35, H50-H65, and H95-H102 according to Kabat numbering, amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of: (i) SEQ ID NO: 1, 2, 3; or (ii) SEQ ID NO: 1, 4, 3.

In one embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof comprises at positions L24-34, L50-56 and L89-97 according to Kabat numbering the amino acid sequences of SEQ ID NO: 5, 6 and 7 for CDR-L1, CDR-L2 and CDR-L3, respectively.

In certain embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof comprises the G55A and G61A mutations in the VH domain according to Kabat numbering. In some embodiments, these mutations reduce the propensity for asparagine deamidation in the anti-ROR1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof with the mutations has increased stability compared to the parent antibody without the mutations.

In some embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but no more than five residue modifications in the CDR sequences of SEQ ID NOS: 1-3 and 5-7. In some embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but no more than five residue modifications in the CDR sequences of SEQ ID NOS: 1, 4, 3 and 5-7. The amino acid modifications can be amino acid substitutions, deletions and/or additions, such as conservative substitutions.

In one embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof of the present invention comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of a heavy chain variable VH domain and a light chain variable VL domain selected from the group consisting of the following VH/VL sequence pairs: SEQ ID NOs: 8/9, 17/9, 10/13, 10/14,10/15, 10/16, 11/13, 11/14, 11/15, 11/16, 12/13, 12/14, 12/15, 12/16 and 21/13. The CDR can be determined by one of skill in the art using the most commonly used CDR determination schemes, such as the Kabat, Chothia or IMGT determination.

In one embodiment, an anti-ROR1 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable VH domain and a light chain variable VL domain, wherein:

- the VH domain comprises the sequence of SEQ ID NO: 8 or 17, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence, and/or

- The VL domain comprises the sequence of SEQ ID NO: 9 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence.

In another embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable VH domain and a light chain variable VL domain, wherein:

- the VH domain comprises a sequence selected from SEQ ID NO: 10-12 and 21, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence, and/or

- The VL domain comprises a sequence selected from SEQ ID NO: 13-16, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence.

In some embodiments, an anti-ROR1 antibody comprises a VH sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, while maintaining the ability to bind to ROR1 with the same or improved binding properties, such as off-rate and/or internalization rate. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted in SEQ ID NO: 8, 17, or SEQ ID NO: 10-12, or 21. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). The anti-ROR1 antibody optionally comprises a VH sequence of SEQ ID NO: 8, 17 or SEQ ID NO: 10-12 or 21, including post-translational modifications of this sequence. In a particular embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2/4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH sequence is a humanized VH sequence.

In some embodiments, an anti-ROR1 antibody comprises a VL sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, while maintaining the ability to bind to ROR1 with the same or improved binding properties, such as off-rate and/or internalization rate. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted in SEQ ID NO: 13. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). An anti-ROR1 antibody optionally comprises the VL sequence of SEQ ID NO: 13, including post-translational modifications of that sequence. In a particular embodiment, the VL sequence comprises one, two, or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VL sequence is a humanized VL sequence.

In one embodiment, an anti-ROR1 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable VH domain comprising or consisting of SEQ ID NO: 21 and a light chain variable VL domain comprising or consisting of SEQ ID NO: 13.

In one embodiment, an isolated anti-ROR1 antibody or antigen-binding fragment of the present invention is a chimeric antibody or a humanized antibody. In some embodiments, the anti-ROR1 antibody or antigen-binding fragment is a humanized antibody.

In some embodiments, a humanized isolated ROR1 antibody or antigen-binding fragment of the present invention comprises one or more backmutations at positions in the framework regions to improve binding properties. In some embodiments, the VH domain of a humanized ROR1 antibody or antigen-binding fragment of the present invention comprises backmutations compared to the human sequence at residues: Glu at position 1 (1E), Tyr at position 27 (27Y), His at position 94 (94H), and optionally one or more of Lys at position 38 (38K), Ile at position 48 (48I), Lys at position 66K, and Ala at position 67 (67A), according to Kabat numbering. In one embodiment, the VL domain of the humanized anti-ROR1 antibody or antigen-binding fragment of the present invention comprises back mutations compared to the human sequence at residues: Tyr at position 71 (71Y) and, optionally, one or more of Leu at position 4 (4L), Arg at position 69 (69R), His at position 49 (49H), He at position 58 (58I) according to Kabat numbering.

In one embodiment, an isolated ROR1 antibody or antigen-binding fragment of the present invention is a humanized antibody comprising backmutated amino acid residues in the VH domain selected from the group consisting of: (i) 1E, 27Y, and 94H, (ii) 1E, 27Y, 48I, 67A, and 94H, (iii) 1E, 27Y, 38K, 481, 67A, 66K, and 94H according to Kabat numbering; and/or backmutated amino acid residues in the VL domain selected from the group consisting of: (i) 71Y; (ii) 49H, 69R and 71Y, (iii) 4L, 69R and 71Y and (iv) 4L, 49H, 581, 69R and 71Y, according to Kabat numbering.

In one embodiment, an isolated anti-ROR1 antibody or antigen-binding fragment of the present invention is a humanized antibody comprising amino acid residues 1E, 27Y, and 94H in the VH domain and amino acid residue 71Y in the VL domain according to Kabat numbering. In a further embodiment, an isolated anti-ROR1 antibody or antigen-binding fragment of the present invention further comprises mutations G55A and G61A in the VH domain according to Kabat numbering.

In some embodiments, an isolated ROR1 antibody or antigen-binding fragment of the present invention comprises a combination of VH and VL sequences selected from the group consisting of:

In some embodiments, said antibody comprises a VH domain comprising or consisting of the sequence of SEQ ID NO: 21 and a VL domain comprising or consisting of the sequence of SEQ ID NO: 13.

In some embodiments of an anti-ROR1 antibody or antigen-binding fragment of the present invention, the antibody or antigen-binding fragment comprises an Fc region, which may be a native or a variant Fc region. In particular embodiments, the Fc region is a human Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to alter (e.g., reduce or eliminate) at least one effector function, such as ADCC and/or CDC. In some embodiments, the present invention provides an anti-ROR1 antibody or antigen-binding fragment comprising an Fc region with one or more mutations to alter at least one effector function, such as L234A and L235A.

In some embodiments, the antigen-binding fragments of the anti-ROR1 antibody of the present invention can be, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; or single-chain antibody molecules (e.g., scFv).

In one embodiment, an anti-ROR1 antibody described herein, or an antigen-binding fragment thereof, binds to an extracellular domain of ROR1 or a fragment thereof. In some embodiments, the extracellular domain of ROR1 comprises the amino acid sequence Q30-Y406 of the human ROR1 protein under UniProt identifier Q01973-1, or the amino acid sequence of SEQ ID NO: 41, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence.

In one embodiment, an anti-ROR1 antibody described herein, or an antigen-binding fragment thereof, binds to ROR1 at the C-terminus of the Ig-like domain of ROR1. In one embodiment, the antibody binds to ROR1 at the same epitope as an antibody with the VH/VL sequence pair of SEQ ID NOs: 8 and 9 (e.g., ROR1-mAb004). In one embodiment, the antibody competes with an antibody with the VH/VL sequence pair of SEQ ID NOs: 42 and 43 (e.g., antibody D10 of WO2012097313) for binding to ROR1.

In one embodiment, the anti-ROR1 antibody described herein or antigen-binding fragment thereof has an association rate constant (k _on ) for human ROR1 of at least 1×10 ⁴ M ^-1 s ^-1 , at least 3×10 ⁴ M ^-1 s ^-1 , at least 5×10 ⁴ M ^-1 s ^-1 , at least 7×10 ⁴ M ^-1 s ^-1 , at least 9×10 ⁴ M ^-1 s ^-1 , at least 1×10 ⁵ M ^-1 s ^-1 , as measured by biolayer interferometry or surface plasmon resonance.

In another embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof described herein has a dissociation rate constant (k _off ) for human ROR1 of less than 5 x 10 ^-3 s ^-1 , less than 3 x 10 x 10 ^-3 s ^-1 , less than 2 x 10 ^-3 s ^-1 , less than 1 x 10 ^-3 s ^-1 , as measured by surface plasmon resonance or biolayer interferometry. In a further embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof described herein is a humanized antibody and has a k _off for human ROR1 of about 1-100%, such as about 3-50%, of the k _{off of} an antibody with the VH/VL sequence pair of SEQ ID NOs: 8 and 9 in the same antibody format for human ROR1. The off-rate can be used to determine the duration of binding of an antibody to its antigen. In general, a higher off-rate value correlates with a slow dissociation of the formed complex, while a lower off-rate value correlates with a fast dissociation. In one embodiment, the anti-ROR1 antibody described herein or antigen-binding fragment thereof has a slower off-rate compared to the off-rate observed for D10 described in WO2012097313 and remains bound to the ROR1 target for a longer time, which may contribute to increased recruitment of effector molecules to ROR1-expressing tumor cells ("ROR1 ⁺ ").

In one embodiment, the anti-ROR1 antibody described herein or antigen-binding fragment thereof has a dissociation constant (K _D ) for ROR1 in the nanomolar (10 ^-7 to 10 ^-9 ) range, such as less than 8 x 10 ^-7 M, less than 5 x 10 ^-7 M, less than 3 x 10 ^-7 M, less than 1 x 10 ^-7 M, less than 8 x 10 ^-8 M, less than 5 x 10 ^-8 M, less than 3 x 10 ^-8 M, less than 2 x 10 ^-8 M, less than 1 x 10 ^-8 M, less than 8 x 10 ^-9 M, less than 6 x 10 ^-9 M, less than 4 x 10 ^-9 M, less than 2 x 10 ^-9 M, or less than 1 x 10 ^-9 M.

In one embodiment, the anti-ROR1 antibody described herein, or an antigen-binding fragment thereof, specifically binds to ROR1 expressed on ROR1 ⁺ target cells, such as CHO cells or ROR1-expressing myeloma cell lines. As measured by flow cytometry in a cell-based assay, the anti-ROR1 antibody exhibits strong binding activity to ROR1 ⁺ cells, which is stronger than the off-rate observed for D10 described in WO2012097313. In some embodiments, the cell binding activity is reflected by the MFI detected at the saturation concentration of the antibody or at an antibody concentration of about 100 nM. In some embodiments, an anti-ROR1 antibody or antigen-binding fragment described herein exhibits higher binding potency to ROR1 expressed on a target cell compared to an antibody with the VH/VL sequence pair of SEQ ID NOs: 44 and 45 (e.g., antibody R12 of WO2014167022) or an antibody binding the same epitope as R12 at the junction of the Ig and Fz domains of ROR1. In one embodiment, the binding activity of the antibody to cells expressing ROR1 is measured using a cell-based assay as described in Example 1.3.

In some embodiments, as expected, an anti-ROR1 antibody described herein with a relatively low affinity for ROR1 in the nanomolar range but strong cell surface binding affinity may promote tumor distribution and/or result in more selective targeting of tumor cells expressing the target at higher density.

In one embodiment, an anti-ROR1 antibody described herein, or an antigen-binding fragment thereof, exhibits minimal internalization when bound to the surface of ROR1-expressing cells. In one embodiment, the internalization rate is no more than 20%, 15%, 14%, 13%, 12%, 11%, or 10%, or the antibody is not internalized, as measured in a cell-based assay. The internalization rate may reflect a decrease in the percent mean fluorescence intensity (MFI), as determined by flow cytometry, upon binding of the antibody to the surface of ROR1-expressing cells after a two-hour incubation at 37°C compared to a control at 4°C for the same period. In one embodiment, internalization of the anti-ROR1 antibody is assayed using a ROR1-expressing myeloma cell line. In one embodiment, prior to detecting MFI, the test antibody is incubated with cells expressing ROR1 for a period of time, such as at 4°C for 30 minutes, to allow binding of the antibody to ROR1 on the cell surface, and then the cells are incubated at 37°C for 2 hours to allow internalization, or maintained at 4°C for the same time as a control. In one embodiment, the rate of internalization is calibrated relative to the rate of internalization measured by incubation at 37°C in the presence of an internalization inhibitor, such as phenylarsine oxide (PAO). In one embodiment, the extent of internalization is measured in a cell-based assay as described in Example 8.

In one embodiment, the antibody may block the binding of ROR1 to its ligand Wnt5a on the surface of cells expressing ROR. In another embodiment, the antibody may be used to inhibit the ROR1/wnt5 signaling pathway. In a further embodiment, the antibody may be used to inhibit the growth and metastasis of cancer associated with the ROR1/wnt5A pathway.

Antibodies against CD3

The present invention also provides antibodies capable of binding human CD3.

In some embodiments, an anti-CD3 antibody of the present invention comprises: a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, wherein:

- CDR-H1 contains the sequence NYYVH (SEQ ID NO: 25);

- CDR-H2 contains the sequence WISPGSDNTKYNEKFKG (SEQ ID NO: 26);

- CDR-H3 contains the sequence DDYGNYYFDY (SEQ ID NO: 27);

- CDR-L1 contains the sequence KSSQSLLNARTRKNYLA (SEQ ID NO: 28);

- CDR-L2 contains the WASTRES sequence (SEQ ID NO: 29);

- CDR-L3 contains the sequence KQSYILRT (SEQ ID NO: 30),

wherein the said CDRs are defined in accordance with the Kabat numbering.

In some embodiments, an anti-CD3 antibody or antigen-binding fragment thereof according to the present application comprises:

- a VH domain comprising the sequence of SEQ ID NO: 22 or 23, or a sequence characterized by at least 80%-90% or 95%-99% sequence identity to said sequence, and/or

- a VL domain comprising the sequence of SEQ ID NO: 24, or a sequence characterized by at least 80%-90% or 95%-99% sequence identity to said sequence.

In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof comprises a VH domain comprising the sequence of SEQ ID NO: 22 and a VL domain comprising the sequence of SEQ ID NO: 24. In other embodiments, the anti-CD3 antibody or antigen-binding fragment thereof comprises a VH domain comprising the sequence of SEQ ID NO: 23 and a VL domain comprising the sequence of SEQ ID NO: 24.

In some embodiments, an anti-ROR1 antibody of the present invention or an anti-CD3 antibody of the present invention can be used to generate derivatives of the binding proteins that recognize the same target antigen using techniques well known in the art. Such a derivative can be, for example, a single chain antibody (scFv), a Fab fragment (Fab), a Fab' fragment, F(ab')2, Fv, and disulfide-linked Fv. Such a derivative can be, for example, a fusion protein or a conjugate comprising an anti-ROR1 antibody of the present invention or an anti-CD3 antibody of the present invention. The fusion protein can be a multispecific antibody or a CAR molecule. The conjugate can be an antibody-drug conjugate (ADC) or an antibody conjugated to a detection agent, such as a radioactive isotope.

Bispecific ROR1×CD3 binding proteins

In another aspect, the present invention provides bispecific ROR1/CD3 binding proteins, particularly "Fab in tandem" immunoglobulins (FIT-Ig) and monovalent asymmetric bispecific tandem Fab antibodies (MAT-Fab) capable of binding to both ROR1 and CD3. Each variable domain (VH or VL) of the FIT-Ig or MAT-Fab may be derived from one or more "parent" monoclonal antibodies that bind one of the target antigens, i.e., ROR1 or CD3. The FIT-Ig or MAT-Fab binding proteins may be prepared using the variable domain sequences of the anti-ROR1 and anti-CD3 monoclonal antibodies described herein. For example, the parent antibodies are humanized antibodies.

One aspect of the present invention relates to selecting parent antibodies having at least one or more properties desirable for a FIT-Ig or MAT-Fab molecule. In one embodiment, the antibody properties are selected from the group consisting of antigen specificity, antigen affinity, dissociation rate, cell binding activity, internalization rate, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding.

In some embodiments, the bispecific FIT-Ig and MAT-Fab proteins of the present invention are configured without the use of an interdomain peptide linker. However, it is common knowledge in the art that adjacent binding sites in multivalent recombinant immunoglobulin formats containing tandem binding sites would interfere with each other unless a flexible linker is used to spatially separate these binding sites. However, for the ROR1/CD3 FIT-Ig and MAT-Fab of the present invention, it has been found that the arrangement of the immunoglobulin domains in accordance with the chain formulas described herein results in polypeptide chains that are well expressed in transfected mammalian cells, assemble properly, and are secreted as bispecific multivalent immunoglobulin-like proteins that bind the ROR1 and CD3 target antigens. See the Examples section below. In addition, the elimination of synthetic linker sequences from the binding proteins avoids the creation of antigenic sites recognized by mammalian immune systems, and thus the elimination of linkers reduces the potential immunogenicity of FIT-Ig and MAT-Fab and results in their half-life in the circulation being similar to that of the natural antibody, i.e. FIT-Ig and MAT-Fab are not rapidly cleared by immune opsonization and hepatic uptake.

In some embodiments, the bispecific ROR1xCD3 binding protein of the present application comprises:

a) the first antigen-binding site that specifically binds ROR1; and

b) a second antigen-binding site that specifically binds CD3.

In one embodiment, the bispecific binding proteins described herein comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, derived from any anti-ROR1 antibody or antigen-binding fragment thereof of the present application and described herein, to form the ROR1 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins described herein comprise a VH/VL pair derived from any anti-ROR1 antibody or antigen-binding fragment thereof of the present application and described herein, to form the ROR1 binding site of the bispecific binding protein.

In one embodiment, the bispecific binding proteins described herein further comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, derived from any anti-CD3 antibody or antigen-binding fragment thereof of the present application and described herein, to form the CD3 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins described herein comprise a VH/VL pair derived from any anti-CD3 antibody or antigen-binding fragment thereof of the present application and described herein, to form the CD3 binding site of the bispecific binding protein.

In one embodiment, the ROR1 binding site and the CD3 binding site in the bispecific ROR1/CD3 binding protein of the present application are humanized, comprising humanized VH/VL sequences, respectively.

Bispecific binding proteins FIT-Ig

In one embodiment, the bispecific ROR1xCD3 binding protein of the present application is a bispecific FIT-Ig binding protein capable of binding ROR1 and CD3. The Fab in tandem immunoglobulin binding protein (FIT-Ig) is a monomeric tetravalent binding protein with dual specificity, comprising six polypeptide chains and four functional Fab binding regions - two external Fab binding regions and two internal Fab binding regions. As shown in Figure 10A, the binding protein adopts the format (outer Fab-inner Fab-Fc) x 2 and binds to both antigen A and antigen B. In one aspect, the bispecific ROR1 x CD3 binding protein of the present application is a bispecific FIT-Ig binding protein, wherein two Fab domains of the FIT-Ig protein form a first antigen-binding site that specifically binds ROR1; and the other two Fab domains of the FIT-Ig protein form a second antigen-binding site that specifically binds CD3. In some embodiments, the FIT-Ig binding protein of the present invention does not utilize a linker between the immunoglobulin domains.

In another embodiment of the present invention, a bispecific Fab in tandem immunoglobulin binding protein (FIT-Ig) is provided, comprising a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein

(i) in the LH format, the first polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL-VH _B -CH1-Fc, wherein CL is fused directly to VH _B ; the second polypeptide chain comprises, from N-terminus to C-terminus, VH _A -CH1; the third polypeptide chain comprises, from N-terminus to C-terminus, VL _B -CL; or

(ii) in the HL format, the first polypeptide chain comprises, from N-terminus to C-terminus, VH _A -CH1-VL _B -CL-Fc, wherein CH1 is fused directly to VL _B ; the second polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL; the third polypeptide chain comprises, from N-terminus to C-terminus, VH _B -CH1;

wherein VL is a variable domain of the light chain, CL is a constant domain of the light chain, VH is a variable domain of the heavy chain, CH1 is a constant domain of the heavy chain, Fc is the Fc region of an immunoglobulin, such as FcIgG1 (e.g., an Fc fragment containing from the N-terminus to the C-terminus a hinge-CH2-CH3),

where VL _A -CL pairs with VH _A -CH1 to form a first Fab that specifically binds a first antigen A, and VL _B -CL pairs with VH _B -CH1 to form a second Fab that specifically binds a second antigen B, and

where the first antigen A is ROR1 and the second antigen B is CD3, or where the first antigen A is CD3 and the second antigen B is ROR1,

wherein two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains associate to form the FIT-Ig binding protein.

In some embodiments of the bispecific FIT-Ig binding protein of the present application, the first polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL-VH _B -CH1-Fc, wherein antigen A is ROR1 and antigen B is CD3, or antigen A is CD3 and antigen B is ROR1.

In some embodiments, a Fab that binds to ROR1 formed by pairing a VL-CL with a VH-CH1 in a FIT-Ig binding protein (e.g., formed by VLA _- CL and VHA _- CH1 when A is ROR1; or formed by _VLB -CL and _VHB -CH1 when B is ROR1) comprises a set of six CDRs, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any ROR1 antibody or antigen-binding fragment thereof of the present application and described herein, to form the ROR1 binding site of the bispecific binding protein. In some further embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprise, respectively, the sequences of SEQ ID NOs: 1, 2, 3 and 5, 6, 7; or the sequences of SEQ ID NOs: 1, 4, 3 and 5, 6, 7.

In some embodiments, a Fab that binds to ROR1 in a FIT-Ig binding protein comprises a VH/VL pair derived from any of the ROR1 antibodies or antigen-binding fragments thereof of the present application and described herein. In some further embodiments, the VH/VL pair comprises sequences selected from the group consisting of the following VH/VL sequence pairs: SEQ ID NO: 8/9, 17/9, 10/13, 10/14, 10/15, 10/16, 11/13, 11/14, 11/15, 11/16, 12/13, 12/14, 12/15, 12/16, and 21/13, or at least 80%, 85%, 90%, 95%, or 99% identical sequences thereto. In some embodiments, a Fab that binds to ROR1 in a FIT-Ig binding protein comprises the VH sequence of SEQ FD NO: 21 and the VL sequence of SEQ ID NO: 13.

In some embodiments, a CD3-binding Fab formed by pairing a VL-CL with a VH-CH1 in a FIT-Ig binding protein (e.g., formed by VLA _- CL and VHA _- CH1 when A is CD3; or formed by _VLB -CL and _VHB -CH1 when B is CD3) comprises a set of six CDRs, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-CD3 antibody or antigen-binding fragment thereof of the present application and described herein, to form a CD3 binding site of the bispecific binding protein. In some embodiments, a Fab that binds to CD3 formed by pairing the VL-CL with the VH-CH1 of a FIT-Ig binding protein comprises a set of six CDRs, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26, 27, and 28, 29, 30, respectively. According to some further embodiments, a Fab that binds to CD3 comprises a VH/VL pair comprising the sequences of SEQ ID NOs: 22 and 24, or sequences that have at least 80%, 85%, 90%, 95%, or 99% sequence identity to these sequences; or sequences of SEQ ID NO: 23 and 24 or sequences that have at least 80%, 85%, 90%, 95% or 99% sequence identity to said sequences.

In another embodiment of the present invention, a bispecific Fab in tandem immunoglobulin binding protein (FIT-Ig) is provided, comprising a first, second and third polypeptide chains,

Where:

(i) in the LH format, the first polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL-VH _B -CH1-Fc, wherein CL is fused directly to VH _B ; the second polypeptide chain comprises, from N-terminus to C-terminus, VH _A -CH1; the third polypeptide chain comprises, from N-terminus to C-terminus, VL _B -CL; or

(ii) in the HL format, the first polypeptide chain comprises, from N-terminus to C-terminus, VH _A -CH1-VL _B -CL-Fc, wherein CH1 is fused directly to VL _B ; the second polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL; the third polypeptide chain comprises, from N-terminus to C-terminus, VH _B -CH1;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, A is an ROR1 epitope and B is a CD3 epitope, or A is a CD3 epitope and B is a ROR1 epitope. According to the present invention, such FIT-Ig binding proteins bind to both ROR1 and CD3.

In some embodiments, the Fab fragments of such FIT-Ig binding proteins comprise VL _A -CL and VH _A -CH1 domains from a parent antibody that binds to one of the ROR1 and CD3 antigens, and VL _B -CL and VH _B -CH1 domains from another parent antibody that binds to the other of the ROR1 and CD3 antigens. In some embodiments, the formation of the VH-CH1::VL-CL pair results in the formation of tandem Fab fragments that recognize both ROR1 and CD3.

According to the present invention, the ROR1/CD3-binding protein FIT-Ig comprises a first, second and third polypeptide chains, wherein the first polypeptide chain comprises from the N-terminus to the C-terminus VL _ROR1 -CL-VH _CD3 -CH1-hinge-CH2-CH3, wherein CL is directly fused to VH _CD3 , wherein the second polypeptide chain comprises from the N-terminus to the C-terminus VH _ROR1 -CH1; and wherein the third polypeptide chain comprises from the N-terminus to the C-terminus VL _CD3 -CL. In alternative embodiments, the ROR1/CD3 binding protein FIT-Ig comprises a first, second, and third polypeptide chain, wherein the first polypeptide chain comprises from N-terminus to C-terminus VH _ROR1 -CH1-VL _CD3 -CL-hinge-CH2-CH3, wherein CH1 is directly fused to VL _CD3 , wherein the second polypeptide chain comprises from N-terminus to C-terminus VL _ROR1 -CL; and wherein the third polypeptide chain comprises from N-terminus to C-terminus VH _CD3 -CH1. In some embodiments, _{the ROR1} VL is a light chain variable domain of an anti-ROR1 antibody, CL is a light chain constant domain, _{the ROR1} VH is a heavy chain variable domain of an anti-ROR1 antibody, CH1 is a heavy chain constant domain, _{the CD3} VL is a light chain variable domain of an anti-CD3 antibody, _{the CD3} VH is a heavy chain variable domain of an anti-CD3 antibody; and, optionally, the _CD3 VL -CL domains are the same as the light chain of the parent anti-CD3 antibody, the _{CD3 VH} -CH1 domains are the same as the heavy chain variable domain and the heavy chain constant domain of the parent anti-CD3 antibody, the _ROR1 VL -CL domains are the same as the light chain of the parent anti-ROR1 antibody, and the _ROR1 VH -CH1 domains are the same as the heavy chain variable domain and the heavy chain constant domain of the parent anti-ROR1 antibody.

In the above formulas for the FIT-Ig binding protein, the Fc region may be a native or variant Fc region. In particular embodiments, the Fc region is a human Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In particular embodiments, the Fc is a human Fc from IgG1 or a modified human Fc comprising one or more mutations to reduce or eliminate at least one effector function of the Fc, such as binding of the Fc to FcγR, ADCC, and/or CDC. The mutations may be, for example, L234A/L235A (numbering according to the Kabat-EC index). In one embodiment, the Fc region is derived from human IgG1 with mutations L234A and L235A, such as those shown below in Table 8 (from AK 104 to AK 227 of SEQ ID NO: 31). In one embodiment, the Fc region comprises the sequence AK 104 - AK 227 of SEQ ID NO: 31 or a sequence that has at least 90%, 95%, 97%, 98%, 99% or more sequence identity to said sequence.

In some embodiments of the FIT-Ig binding protein of the present invention, the CH1, CL and Fc domains are human sequences or are derived from them. In some embodiments of the FIT-Ig binding protein of the present invention, CH1 is a human IgG1 CH1 constant domain, such as having the sequence of SEQ ID NO: 33, or a sequence that has at least 90%, 95%, 97%, 98%, 99% or more sequence identity to this sequence. In the above formulas for the FIT-Ig binding protein, CL is a human CL kappa constant domain, such as having the sequence of SEQ ID NO: 32, or a sequence that has at least 90%, 95%, 97%, 98%, 99% or more sequence identity to this sequence.

In one embodiment, the FIT-Ig binding proteins of the present invention retain one or more properties of the parent antibodies. In some embodiments, the FIT-Ig retains a binding affinity for target antigens (i.e., CD3 and ROR1) comparable to the binding affinity of the parent antibodies, meaning that the binding affinity of the FIT-Ig for the target antigens ROR1 and CD3 is changed by no more than 10-fold compared to the binding affinity of the parent antibodies for the corresponding target antigens, as measured by surface plasmon resonance or biolayer interferometry.

In one embodiment, the FIT-Ig binding protein of the present invention binds ROR1 and CD3 and consists of a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein:

- the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34 or 37 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence,

- the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence, and

- the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence.

In one embodiment, the FIT-Ig binding protein of the present invention binds ROR1 and CD3 and consists of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 34 or 37; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 35; and a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 36.

Bispecific binding protein MAT-Fab

In one embodiment, the bispecific ROR1xCO3 binding protein of the present application is a MAT-Fab bispecific binding protein capable of binding ROR1 and CD3. The monovalent asymmetric tandem Fab (MAT-Fab) bispecific binding protein is a monomeric bivalent binding protein with dual specificity comprising four polypeptide chains and two functional Fab binding regions in tandem. As shown in Figure 10B, the binding protein uses an outer Fab-inner Fab dimer Fc:Fc format and binds both antigen A and antigen B. In some embodiments, the bispecific ROR1xCD3 binding protein of the present application is a MAT-Fab bispecific binding protein, wherein one Fab domain of the MAT-Fab protein forms a first antigen-binding site that specifically binds ROR1; and the other Fab domain of the MAT-Fab protein forms a second antigen-binding site that specifically binds CD3.

In a further embodiment of the present invention, a bispecific binding protein with a monovalent asymmetric tandem Fab (MAT-Fab) is provided, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain, wherein

(i) in the LH format, the first polypeptide chain comprises from N-terminus to C-terminus VL _A -CL-VH _B -CH1-Fc, wherein CL is fused directly to VH _B ; the second polypeptide chain comprises from N-terminus to C-terminus VH _A -CH1; the third polypeptide chain comprises from N-terminus to C-terminus VL _B -CL; and the fourth polypeptide chain comprises Fc; or

(ii) in the FTL format, the first polypeptide chain comprises, from N-terminus to C-terminus, VH _A -CH1-VL _B -CL-Fc, wherein CH1 is attached directly to VL _B ; the second polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL; the third polypeptide chain comprises, from N-terminus to C-terminus, VH _B -CH1; and the fourth polypeptide chain comprises Fc;

wherein VL is a variable domain of a light chain, CL is a constant domain of a light chain, VH is a variable domain of a heavy chain, CH1 is a constant domain of a heavy chain, Fc is an Fc region of an immunoglobulin, such as Fc of IgG1 (e.g., an Fc fragment comprising from the N-terminus to the C-terminus a hinge-CH2-CH3),

where VL _A -CL pairs with VH _A -CH1 to form a first Fab that specifically binds a first antigen A, and VL _B -CL pairs with VH _B -CH1 to form a second Fab that specifically binds a second antigen B, and

where the first antigen A is ROR1 and the second antigen B is CD3, or where the first antigen A is CD3 and the second antigen B is ROR1,

wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain associate to form a MAT-Fab binding protein.

In some embodiments of the MAT-Fab binding protein of the present invention, the Fc is an immunoglobulin Fc region comprising, from the N-terminus to the C-terminus, a hinge-CH2-CH3, wherein the hinge-CH2 is a hinge-CH2 region of an immunoglobulin heavy chain, wherein the hinge-CH2 region is fused directly to CH3, and wherein the Fc region of the first polypeptide chain comprises a first CH3 domain (a CH3m1 domain), and the Fc region of the fourth polypeptide chain comprises a second CH3 domain (a CH3m2 domain). In further embodiments, the Fc regions of the first and fourth polypeptide chains, particularly in their CH3 domains, comprise heterodimerizing modifications that promote heterodimerization over homodimerization of the two Fc regions. In some embodiments, knob-into-hole heterodimerization technology is used to promote chain heterodimerization. The MAT-Fab binding protein optionally further comprises a mutation in the first CH3 domain (the CH3m1 domain) and in the second CH3 domain (the CH3m2 domain) resulting in the introduction of a cysteine residue that facilitates the formation of a disulfide bond when pairing the two said CH3 domains.

In some embodiments, one or more knob-into-hole (KiH) mutations are introduced into the first CH3 domain (CH3m1 domain) of the first chain and the second CH3 domain (CH3m2 domain) of the fourth chain. In a further embodiment, when the first CH3 domain (CH3m1 domain) of the first chain is mutated to form a structural knob, the second CH3 domain (CH3m2 domain) of the fourth chain is mutated to form a complementary structural hole, which facilitates pairing of the first CH3 domain with the second CH3 domain; or when the first CH3 domain (CH3m1 domain) of the first chain is mutated to form a structural hole, the second CH3 domain (CH3m2 domain) of the fourth chain is mutated to form a complementary structural knob, which facilitates pairing of the first CH3 domain with the second CH3 domain. In some embodiments, the knob mutation is a T366W substitution and the complementary trough mutations are T366S, L368A, and Y407V substitutions.

In some embodiments, the bispecific binding protein of the present invention is a MAT-Fab protein with a typical knob substitution (T366W) in the first CH3 domain and a corresponding hole substitution (T366S, L368A and Y407V) in the second CH3 domain and optionally with two additional inserted cysteine residues S354C/Y349C (contained in the corresponding CH3 sequences). For example, the first CH3 domain (CH3m1 domain) may comprise a T366W knob substitution and an inserted cysteine residue S354C, and the second CH3 domain (CH3m2 domain) comprises T366S, L368A and Y407V as hole substitutions and an inserted cysteine residue Y349C.

Knob-into-hole dimerization modules and their application in antibody engineering are well known in the art and are described, for example, by Ridgway et al., 1996, Protein Engineering 9(7) 617-621. The introduction of an additional disulfide bridge into the CH3 domain is reported, for example, by Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681.

In some embodiments of the bispecific binding protein MAT-Fab according to the present application, the first polypeptide chain comprises from the N-terminus to the C-terminus VL _A -CL-VH _B -CH1-Fc, wherein antigen A is ROR1, antigen B is CD3, or antigen A is CD3, antigen B is ROR1.

In some embodiments, a Fab that binds to ROR1 formed by pairing VL-CL with VH-CH1 in a MAT-Fab binding protein (e.g., formed by VLA _- CL and VHA _- CH1 when A is ROR1) comprises a set of six CDRs, namely, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any ROR1 antibody or antigen-binding fragment thereof of the present application and described herein, to form the ROR1 binding site of the bispecific binding protein. In some embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 1, 2, 3 and 5, 6, 7, respectively; or comprise the sequences of SEQ ID NO: 1, 4, 3 and 5, 6, 7, respectively. In some embodiments, the Fab that binds to ROR1 in the MAT-Fab binding protein comprises a VH/VL pair derived from any ROR1 antibody or antigen-binding fragment thereof according to the present application and described herein, to form an ROR1 binding site of the bispecific binding protein. In some embodiments, the VH/VL pair comprises sequences selected from the group consisting of the following VH/VL sequence pairs: SEQ ID NO: 8/9, 17/9, 10/13, 10/14, 10/15, 10/16, 11/13, 11/14, 11/15, 11/16, 12/13, 12/14, 12/15,12/16, and 21/13, or sequences that are at least 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, a Fab that binds to ROR1 in a MAT-Fab binding protein comprises a VH sequence of SEQ ID NO: 21 and a VL sequence of SEQ ID NO: 13.

In some embodiments, a CD3-binding Fab formed by pairing VL-CL with VH-CH1 in a MAT-Fab binding protein (e.g., formed by VL _B -CL and VH _B -CH1 when B is CD3) comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-CD3 antibody or antigen-binding fragment thereof of the present application and described herein, to form a CD3 binding site of the bispecific binding protein. In some embodiments, a Fab that binds to CD3 formed by pairing the VL-CL with the VH-CH1 in a MAT-Fab binding protein comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, comprising the sequences of SEQ ID NOs: 25, 26, 27 and 28, 29, 30, respectively. In some further embodiments, a Fab that binds to CD3 comprises a VH/VL pair comprising the sequences of SEQ ID NOs: 22 and 24 or sequences at least 80%, 85%, 90%, 95% or 99% identical thereto; or the sequences of SEQ ID NOs: 23 and 24 or sequences at least 80%, 85%, 90%, 95% or 99% identical thereto.

In a further embodiment of the present invention, a bispecific binding protein with a monovalent asymmetric tandem Fab (MAT-Fab) is provided, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain, wherein

(i) in the LH format, the first polypeptide chain comprises from N-terminus to C-terminus VL _A -CL-VH _B -CH1-Fc, wherein CL is directly fused to VH _B ; the second polypeptide chain comprises from N-terminus to C-terminus VH _A -CH1; the third polypeptide chain comprises from N-terminus to C-terminus VL _B -CL; and the fourth polypeptide chain comprises Fc; or

(ii) in the HL format, the first polypeptide chain comprises, from N-terminus to C-terminus, VHA- _CH1 - _VLB -CL-Fc, where CH1 is fused directly to _VLB ; the second polypeptide chain comprises, from N-terminus to C-terminus, _VLA -CL; the third polypeptide chain comprises, from N-terminus to C-terminus, VHr-CH1; and the fourth polypeptide chain comprises Fc;

wherein VL is a variable domain of a light chain, CL is a constant domain of a light chain, VH is a variable domain of a heavy chain, CH1 is a constant domain of a heavy chain, Fc is an Fc region of an immunoglobulin comprising from the N-terminus to the C-terminus a hinge-CH2-CH3, A is an epitope of ROR1 and B is an epitope of CD3, or A is an epitope of CD3 and B is an epitope of ROR1. According to the present invention, such MAT-Fab binding proteins bind to both ROR1 and CD3.

In some embodiments, the Fab fragments of such MAT-Fab binding proteins comprise _VLA -CL and VHA _- CH1 domains from a parent antibody that binds to one of the ROR1 and CD3 antigens (e.g., an anti-ROR1 antibody or an anti-CD3 antibody described herein) and _VLB -CL and _VHLB -CH1 domains from another parent antibody that binds to the other of the ROR1 and CD3 antigens (e.g., an anti-ROR1 antibody or an anti-CD3 antibody described herein). In some embodiments, formation of the VH-CH1::VL-CL pair results in the formation of tandem Fab fragments that recognize both ROR1 and CD3.

According to the present invention, the ROR1/CD3-binding protein MAT-Fab comprises a first, second, third and fourth polypeptide chains, wherein the first polypeptide chain comprises from the N-terminus to the C-terminus VL _ROR1 -CL-VH _CD3 -CH1- hinge-CH2-CH3m1 , wherein CL is fused directly to VH _CD3 ; wherein the second polypeptide chain comprises from the N-terminus to the C-terminus VH _ROR1 -CH1; wherein the third polypeptide chain comprises from the N-terminus to the C-terminus VL _CD3 -CL; and wherein the fourth polypeptide chain is an Fc polypeptide chain comprising n-terminus-CH2-CH3m2. In alternative embodiments, the ROR1/CD3 binding protein MAT-Fab comprises a first, second, third, and fourth polypeptide chains, wherein the first polypeptide chain comprises from N-terminus to C-terminus VH _ROR1 -CH1-VL _CD3 -CL- hinge-CH2-CH3m1 , wherein CH1 is directly fused to VL _CD3 ; wherein the second polypeptide chain comprises from N-terminus to C-terminus VL _ROR1 -CL; wherein the third polypeptide chain comprises from N-terminus to C-terminus VH _CD3 -CH1; and wherein the fourth polypeptide chain is an Fc polypeptide chain comprising hinge-CH2-CH3m2. In some embodiments, the VL _{of ROR1} is the variable domain of a light chain of an anti-ROR1 antibody, CL is the constant domain of a light chain, the VH _{of ROR1} is the variable domain of a heavy chain of an anti-ROR1 antibody, CH1 is the constant domain of a heavy chain, the VL _{of CD3} is the variable domain of a light chain of an anti-CD3 antibody, the VH _{of CD3} is the variable domain of a heavy chain of an anti-CD3 antibody, and one or more knob-into-hole mutations are introduced into the CH3m1 and CH3m2 domains to promote heterodimerization of the CH3m1 and CH3m2 domains; and the VL _CD3 -CL domains are the same as the light chain of the parent anti-CD3 antibody, the VH _CD3 -CH1 domains are the same as the variable and constant domains of the heavy chain of the parent anti-CD3 antibody, the VL _ROR1 -CL domains are the same as the light chain of the parent anti-ROR1 antibody, and the VH _ROR1 -CH1 domains are the same as the variable and constant domains of the heavy chain of the parent anti-ROR1 antibody.

In the above formulas for the first polypeptide chain of the MAT-Fab binding protein, the Fc region may be a native or a variant Fc region. In particular embodiments, the Fc region is a human Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In particular embodiments, the Fc is a human IgG Fc or a variant thereof. In some embodiments, the Fc region is a variant Fc region comprising mutations to reduce or eliminate at least one effector function of the Fc region, such as binding of the Fc to FcγR, ADCC, and/or CDC. The mutations may be, for example, L234A and L235A (numbering according to the EU Kabat index). In one embodiment, said Fc region is a human IgG1 region with the L234A and L235A mutations.

In some embodiments of the MAT-Fab binding protein of the present invention, the CH1, CL and Fc domains are or are derived from human sequences. In some embodiments of the MAT-Fab binding protein of the present invention, CH1 is a heavy chain constant domain, such as a human IgG1 CH1 constant domain, such as having the sequence of SEQ ID NO: 33, or a sequence that has at least 90%, 95%, 97%, 98%, 99% or more sequence identity to this sequence. In the above formulas for the MAT-Fab binding protein, CL is a light chain constant domain, such as a human CL kappa constant domain, such as having the sequence of SEQ ID NO: 32, or a sequence that has at least 90%, 95%, 97%, 98%, 99% or more sequence identity to this sequence.

In some embodiments, the MAT-Fab binding protein of the present invention does not utilize a linker between immunoglobulin domains.

According to one embodiment, the MAT-Fab binding proteins of the present invention retain one or more properties of the parent antibodies. In some embodiments, the MAT-Fab retains a binding affinity for target antigens (i.e., CD3 and ROR1) comparable to the binding affinity of the parent antibodies, meaning that the binding affinity of the MAT-Fab for the target antigens ROR1 and CD3 is changed by no more than 10-fold compared to the binding affinity of the parent antibodies for the corresponding target antigens, as measured by surface plasmon resonance or biolayer interferometry.

In one embodiment, the MAT-Fab binding protein of the present invention binds ROR1 and CD3 and consists of a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide, wherein:

- the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38 or 40 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence,

- the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with respect to said sequence,

- the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence; and

- the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39 or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said sequence.

In one embodiment, the MAT-Fab binding protein of the present invention binds ROR1 and CD3 and consists of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 38 or 40; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 35; a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 36; and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39.

Properties of bispecific binding proteins

In one embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or MAT-Fab capable of binding both CD3 and ROR1 described herein comprises a humanized ROR binding site or a chimeric ROR1 binding site, such as a humanized ROR binding site. In one embodiment, the humanized ROR1 binding site in the FIT-Ig or MAT-Fab protein format has a reduced off rate of ROR1 binding compared to a chimeric ROR1 binding site in the same FIT-Ig or MAT-Fab format consisting of the VH and VL pair of SEQ ID NOs: 8 and 9. In a further embodiment, the off rate ratio of the humanized ROR1 binding site compared to the chimeric ROR1 binding site is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, as measured by surface plasmon resonance or biolayer interferometry. In one embodiment, the dissociation rate of the FIT-Ig binding protein described herein with respect to ROR1 is less than 2×10 ^-3 s ^-1 , 1×10 ^-3 s ^-1 , 8×10 ^-4 s ^-1 , 6×10 ^-4 s ^-1 , 5×10 ^-4 s ^-1 , 4×10 ^-4 s ^-1 , 3×10 ^-4 s ^-1 , 2×10 ^-4 s ^-1 , 1×10 ^-4 s ^-1 , 8×10 ^-5 s ^-1 , 6×10 ^-5 s ^-1 , as measured by surface plasmon resonance or biolayer interferometry. In one embodiment, the FIT-Ig antibody binding protein described herein, or an antigen-binding fragment thereof, has a dissociation constant (K _D ) for ROR1 in the range of 10 ^-8 to 10 ^-10 , such as less than 8 x 10 ^-8 M, less than 5 x 10 ^-8 M, less than 3 x 10 ^-8 M, less than 2 x 10 ^-8 M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-8 M, less than 8 x 10 ^-9 M, less than 6 x 10 ^-9 M, less than 4 x 10 ^-9 M, less than 2 x 10 ^-9 M, or less than 1 x 10 ^-9 M, less than 8 x 10 ^-10 M, less than 6 x 10 ^-10 M, less than 4 x 10 ^-10 M, less than 2 x 10 ^-10 M, or less 1×10 ^-10 M. In one embodiment, the FIT-Ig antibody binding protein described herein, or an antigen-binding fragment thereof, has a dissociation rate for ROR1 binding in the range of 1×10 ^-3 s ^-1 to 1×10 ^-4 s ^-1 , such as less than 2×10 ^-4 s ^-1 , and a K _D in the range of 1×10 ^-9 s ^-1 to 1×10 ^-10 s ^-1 , such as less than 6×10 ^-10 s ^-1 .

In one embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or the binding protein MAT-Fab capable of binding CD3 and ROR1 described herein can be expressed in cultures of transfected mammalian host cells, such as CHO cells or HEK293 cells, at concentrations exceeding 10 mg of the ROR1/CD3 binding protein FIT-Ig or the binding protein MAT-Fab per liter of cell culture (>10 mg/L). In one embodiment, the expression level of the binding protein FIT-Ig or MAT-Fab is greater than 15 mg/L, such as from 15 mg/L to 100 mg/L or more. In another embodiment, the expression level of the binding protein FIT-Ig or MAT-Fab is greater than 20 mg/L.

In one embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or the MAT-Fab binding protein capable of binding CD3 and ROR1 described herein, after single-step purification from cell culture medium using Protein A affinity chromatography, is characterized by a purity of at least 90% as determined by SEC-HPLC. In one embodiment, the binding proteins subjected to the single-step purification are characterized by a purity of at least 91%, 92%, 93%, 95%, 97%, 99% as determined by SEC-HPLC.

In one embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or the binding protein MAT-Fab described herein exhibits minimal internalization upon cell binding to the surface of ROR1-expressing cells. In one embodiment, the internalization rate is no more than 20%, 15%, 14%, 13%, 12%, 11%, 10%, or the binding protein is not internalized, as determined by a cell-based assay.

In one embodiment, the bispecific ROR1/CD3 binding protein or MAT-Fab binding protein described herein is capable of binding both CD3-expressing cells and ROR1-expressing cells. In one embodiment, the CD3-expressing cells are CHO cells transfected with a human TCR/CD3 complex or human T cells. In one embodiment, the ROR1-expressing cells are tumor cells expressing ROR1, such as human non-small cell lung cancer cells, human breast cancer cells, lung carcinoma cells, or myeloma cells.

In one embodiment, the binding efficiency of the FIT-Ig bispecific binding protein to ROR1-expressing cells, as measured by flow cytometry in a cell-based assay, is equivalent to or comparable to the efficiency of the corresponding parental anti-ROR1 monoclonal IgG antibody comprising the same VH/VL sequence pairs for binding ROR1 as the FIT-Ig bispecific protein. In one embodiment, the binding efficiency of the FIT-Ig bispecific binding protein to CD3-expressing cells, as measured by flow cytometry, such as in the assay described in Example 4, is equivalent to or relatively lower (but no more than 10-fold, such as no more than 2-fold, 1-fold, or 50% lower) than the efficiency of the corresponding parental anti-CD3 monoclonal antibody comprising the same VH/VL sequence pairs for binding CD3 as the bispecific binding protein.

In one embodiment, a bispecific binding protein described herein can modulate the biological function of ROR1 and/or CD3. In one embodiment, a bispecific ROR1/CD3 binding protein FIT-Ig or a MAT-Fab binding protein described herein can activate the CD3 signaling pathway in a ROR1-dependent manner. In one embodiment, the bispecific binding proteins of the present disclosure exhibit ROR1-dependent T cell activation. In one embodiment, a bispecific ROR1/CD3 binding protein FIT-Ig or a MAT-Fab binding protein described herein exhibits ROR1-redirected T cell cytotoxicity. In one embodiment, the bispecific binding proteins of the present invention are used to redirect T cell cytotoxic activity toward ROR1-expressing cells without MHC limitations.

In one embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or the binding protein MAT-Fab described herein exhibits ROR1-dependent CD3 activation. In one embodiment, upon binding to ROR1-expressing cells, the ROR1/CD3 bispecific antibodies induce cross-linking of the CD3/TCR complex on T cells and activation of the CD3 signaling pathway. In one embodiment, the ratio of ROR1-expressing target cells to effector T cells is approximately 1:1. In a further embodiment of the invention, the bispecific ROR1/CD3 binding proteins exhibit enhanced T cell activation in the presence of ROR1-expressing target cells and significantly weaker non-specific CD3 redirected activation in the absence of ROR1-expressing target cells compared to the corresponding parental CD3 monoclonal antibodies comprising the same VH/VL sequence pairs for CD3 binding as the bispecific FIT-Ig or MAT-Fab proteins, e.g., as measured at a target cell to effector T cell ratio of approximately 1:1.

In one embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or the binding protein MAT-Fab described herein redirects T cell cytotoxicity to tumor cells expressing ROR1. In another embodiment, the bispecific ROR1/CD3 binding protein FIT-Ig or the binding protein MAT-Fab described herein exhibits anti-tumor activity, such as reducing tumor burden, inhibiting tumor growth, or suppressing tumor cell proliferation.

Pharmaceutical compositions

The present invention also provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof, or a bispecific multivalent binding protein of the present invention (i.e., the primary active ingredient) and a pharmaceutically acceptable carrier. In a specific embodiment, said composition comprises one or more of the antibodies or binding proteins of the present invention. The present invention also provides pharmaceutical compositions comprising a combination of the anti-ROR1 and anti-CD3 antibodies described herein, or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier. In particular, the present invention provides pharmaceutical compositions comprising at least one FIT-Ig binding protein capable of binding ROR1 and CD3, and a pharmaceutically acceptable carrier. In particular, the present invention provides pharmaceutical compositions comprising at least one MAT-Fab binding protein capable of binding ROR1 and CD3, and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may further comprise at least one additional active ingredient. In some embodiments, such an additional ingredient includes a prophylactic and/or therapeutic agent, a detection agent, such as, but not limited to, an antineoplastic drug, a cytotoxic agent, an antibody with a different specificity or a functional fragment thereof, a detectable label or a reporter. In one embodiment, the pharmaceutical composition comprises one or more additional prophylactic or therapeutic agents, i.e., agents other than the antibodies or binding proteins of the present invention, for the treatment of a disorder in which ROR1 activity is detrimental. In one embodiment, the additional prophylactic or therapeutic agents are known to be useful, have been used, or are currently used to prevent, treat, control, or alleviate the disorder or one or more symptoms thereof.

Pharmaceutical compositions comprising the proteins of the present invention are intended, inter alia, for use in diagnosing, detecting or monitoring a disorder, treating, managing or ameliorating a disorder or one or more symptoms thereof; and/or for research purposes. In some embodiments, the composition may also contain a carrier, diluent or excipient. An excipient is generally any compound or combination of compounds that imparts a desirable property to the composition other than that of the principal active ingredient (i.e., other than an antibody, a functional fragment thereof or a binding protein of the present invention).

Nucleic acid, vector and host cells

In another aspect, the present invention provides isolated nucleic acids encoding one or more amino acid sequences of an anti-ROR1 antibody of the present invention or an antigen-binding fragment thereof; isolated nucleic acids encoding one or more amino acid sequences of an anti-CD3 antibody of the present invention or an antigen-binding fragment thereof; and isolated nucleic acids encoding one or more amino acid sequences of a bispecific binding protein comprising a Fab immunoglobulin in tandem (FIT-Ig) and a MAT-Fab binding protein capable of binding both ROR1 and CD3. Such nucleic acids can be inserted into a vector to perform various genetic assays or to express, investigate, or improve one or more properties of an antibody or binding protein described herein. The vector may comprise one or more nucleic acid molecules encoding one or more of the amino acid sequences of the antibody or binding protein described herein, wherein said one or more nucleic acid molecules are operably linked to appropriate transcriptional and/or translational sequences that provide for expression of the antibody or binding protein in a particular host cell carrying the vector. Examples of vectors for cloning or expressing nucleic acids encoding the amino acid sequences of the binding proteins described herein include, but are not limited to, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ, and derivatives thereof.

The present invention also provides a host cell expressing or capable of expressing a vector comprising a nucleic acid encoding one or more of the amino acid sequences of an antibody or binding protein described herein. Host cells that can be used in the present invention can be prokaryotic or eukaryotic. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells that can be used as host cells according to the present invention include protozoan cells, animal cells, plant cells and fungal cells. An exemplary fungal cell is a yeast cell, including Saccharomyces cerevisiae. An exemplary animal cell that can be used as a host cell according to the present invention includes, but is not limited to, a mammalian cell, an avian cell and an insect cell. Exemplary mammalian cells include, but are not limited to, CHO cells, HEK cells and COS cells.

Production methods

In another aspect of the present invention, there is provided a method for producing an antibody against ROR1 or a functional fragment thereof, comprising culturing a host cell containing an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient for the host cell to express the antibody or fragment capable of binding ROR1.

In another aspect of the present invention, there is provided a method for producing an anti-CD3 antibody or a functional fragment thereof, comprising culturing a host cell containing an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient for the host cell to express the antibody or fragment capable of binding CD3.

In another aspect of the present invention, there is provided a method for producing a bispecific multivalent binding protein capable of binding ROR1 and CD3, in particular a FIT-Ig or MAT-Fab binding protein that binds ROR1 and CD3, comprising culturing a host cell comprising an expression vector encoding the FIT-Ig or MAT-Fab binding protein in a culture medium under conditions sufficient for the host cell to express the binding protein capable of binding ROR1 and CD3. The proteins produced by the methods described herein can be isolated and used in the various compositions and methods described herein.

Application of antibodies and binding proteins

Given the ability of the antibodies described herein, functional fragments thereof and bispecific multivalent binding proteins described herein to bind to human ROR1 and/or CD3, they can be used to detect ROR1 and/or CD3, for example, in a biological sample containing cells expressing one or both of these target antigens. The antibodies, functional fragments and binding proteins of the present invention can be used in a conventional immunoassay, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) or an immunohistochemical analysis of tissues. The present invention provides a method for detecting ROR1 or CD3 in a biological sample, comprising contacting the biological sample with an antibody, an antigen-binding fragment thereof or a binding protein of the present invention and detecting the presence or absence of binding to the target antigen, which allows detecting the presence or absence of the target in the biological sample. The antibody, functional fragment or binding protein may be directly or indirectly labeled with a detectable compound to facilitate detection of the bound or unbound antibody/fragment/binding protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive materials include ^3H , ^14C , ^35S , ^90Y , ^99Tc , ^111In , ^125I , ^131I , ^177Lu , ^166Ho , or ^153Sm .

In some embodiments, the antibodies, functional fragments thereof according to the present invention are capable of neutralizing human ROR1 activity both in vitro and in vivo. Accordingly, the antibodies, functional fragments thereof according to the present invention can be used to inhibit human ROR1 activity, for example, inhibit signaling pathways mediated by ROR1, in a cell culture containing ROR1-expressing cells, in human subjects or in other mammals that have ROR1, with which the antibody, functional fragment thereof or binding protein according to the present invention interacts.

In another embodiment, the present invention provides an antibody or bispecific binding protein of the present invention for use in the treatment of a subject suffering from a disease or disorder in which ROR1 activity is detrimental, wherein said antibody or binding protein is administered to said subject, resulting in a decrease in ROR1-mediated activity in said subject. As used herein, the term "disorder in which ROR1 activity is detrimental" is intended to include diseases and other disorders in which the interaction of ROR1 with its ligand (Wnt-5A) in a subject suffering from the disorder is responsible for the pathophysiology of the disorder or is a factor contributing to the worsening of the disorder. Accordingly, a disorder in which ROR1 activity is detrimental is a disorder in which inhibition of ROR1 activity is expected to alleviate the symptoms and/or progression of the disorder. In one embodiment, an anti-ROR1 antibody, a functional fragment thereof, according to the present invention is used in a method for inhibiting the growth or survival of malignant cells or reducing tumor burden.

In some embodiments, the bispecific binding proteins (FIT-Ig or MAT-Fab) of the present invention are capable of redirecting T cell cytotoxicity to ROR-expressing cells both in vitro and in vivo. Accordingly, the bispecific binding proteins of the present invention can be used to inhibit the growth or expansion of ROR1-expressing cancer cells in human subjects or other mammalian subjects that have ROR1 with which an antibody, a functional fragment thereof, or a bispecific binding protein of the present invention cross-reacts.

In another embodiment of the present invention, there is provided a bispecific CD3/ROR1 binding protein (FIT-Ig or MAT-Fab) for use in the treatment of a malignancy expressing ROR1 in a subject, wherein said binding protein is administered to said subject. In some embodiments, the malignancy is a solid tumor or a hematopoietic malignancy.

The antibodies (including functional fragments thereof) and binding proteins of the present invention can be included in pharmaceutical compositions suitable for administration to a subject. Typically, a pharmaceutical composition comprises an antibody or binding protein of the present invention and a pharmaceutically acceptable carrier. In the present application, the term "pharmaceutically acceptable carrier" includes all kinds of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and agents delaying absorption, and the like physiologically compatible substances. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it is preferable to include isotonic agents in the composition, for example, carbohydrates, polyhydric alcohols (e.g., mannitol or sorbitol), or sodium chloride. Pharmaceutically acceptable carriers may additionally contain minor amounts of auxiliary substances, such as humectants or emulsifiers, preservatives or buffers that increase the shelf life or effectiveness of the antibody or binding protein present in the composition. The pharmaceutical composition according to the present invention is formulated so that it is compatible with the intended route of administration.

The method of the present invention may comprise administering a composition formulated for parenteral administration by injection (e.g. by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g. in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous carrier media, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the principal active ingredient may be in powder form for reconstitution with a suitable carrier medium (e.g. sterile, pyrogen-free water) before use.

Use of the present invention may include administration of compositions formulated as depot preparations. Such long-acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. For example, compositions may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g. as a sparingly soluble salt).

The antibody, functional fragment thereof or binding protein of the present invention can also be administered with one or more additional therapeutic agents useful for treating various diseases. The antibodies, functional fragments thereof and binding proteins described in the present application can be used alone or in combination with an additional agent, for example, an additional therapeutic agent, wherein the additional agent is selected by a person skilled in the art according to its purpose. For example, the additional agent can be a therapeutic agent recognized in the art as useful for treating a disease or condition treated by the antibody or binding protein of the present invention. The additional agent can also be an agent that imparts a beneficial property to the therapeutic composition, for example, an agent that affects the viscosity of the composition.

Treatment methods and medical applications

In one embodiment, the present invention provides methods of treating a disorder in which ROR1-mediated signaling pathway activity is associated with or detrimental (e.g., ROR ⁺ solid tumors or hematopoietic malignancies) in a subject in need thereof, said method comprising administering to said subject an anti-ROR1 antibody or ROR1-binding fragment thereof described herein, said antibody or binding fragment capable of binding ROR1 and inhibiting ROR1-mediated signaling in a cell expressing ROR1. In another embodiment, the present invention provides the use of an effective amount of an anti-ROR1 antibody or antigen-binding fragment thereof described herein in treating such a disorder. In another embodiment, the present invention provides the use of an anti-ROR1 antibody or antigen-binding fragment thereof described herein for the preparation of a composition for treating such a disorder. In another embodiment, the present invention provides an anti-ROR1 antibody or antigen-binding fragment thereof described herein for use in the treatment of such a disorder.

In a further embodiment of the method or use described herein, the anti-ROR1 antibody or antigen-binding fragment of the present invention binds ROR1 and comprises a VH domain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 10 or 21, and a VL domain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 13.

In another embodiment, the present invention provides methods for treating a disorder in which ROR1-mediated signaling pathway activity is associated with or deleterious (e.g., ROR ⁺ solid tumors or hematopoietic malignancies) in a subject in need thereof, said method comprising administering to said subject a bispecific FIT-Ig or MAT-Fab binding protein capable of binding CD3 and ROR1 described herein, wherein the binding protein is capable of binding CD3 and ROR1 and inducing redirected T cell cytotoxicity to tumor cells expressing ROR1. In another embodiment, the present invention provides the use of an effective amount of a bispecific FIT-Ig or MAT-Fab binding protein described herein for treating such a disorder. In another embodiment of the present invention, there is provided the use of the bispecific FIT-Ig or MAT-Fab binding protein described herein for the preparation of a composition for the treatment of such a disorder. In another embodiment of the present invention, there is provided the bispecific FIT-Ig or MAT-Fab binding protein described herein for use in the treatment of such a disorder.

In another embodiment of the method or use described herein, the FIT-Ig binding protein of the present invention binds ROR1 and CD3 and consists of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 34 or 37; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 35; and a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 36. In a further embodiment, the MAT-Fab binding protein of the present invention binds ROR1 and CD3 and consists of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 38 or 40; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 35; a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 36; and a fourth polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 39.

In some embodiments, disorders that can be treated with an antibody or binding protein of the present invention include various hematopoietic and solid malignancies that express ROR1 on the surface of malignant cells. In another embodiment, the antibody or binding protein inhibits the growth or survival of malignant cells. In another embodiment, the antibody or binding protein reduces tumor burden. In another embodiment, the cancer is breast cancer, such as triple-negative adenocarcinoma of the breast, or leukemia, such as chronic lymphocytic leukemia (CLL).

The methods of treatment described in the present application may further comprise administering to a subject in need thereof an additional active ingredient suitably present in combination with the present antibody or binding protein for the intended purposes of treatment, for example another drug having antitumor activity. In the method of treatment according to the present invention, the additional active ingredient may be included in a composition comprising an antibody or binding protein according to the present invention and the composition administered to a subject in need of treatment. In another embodiment, the method of treatment according to the present invention may comprise the step of administering to a subject in need of treatment an antibody or binding protein described in the present application and a separate step of administering the additional active ingredient to the subject before, simultaneously or after the step of administering to said subject the antibody or binding protein according to the present invention.

Having now described the present invention in detail, the description will be more fully understood by reference to the following examples, which are included for illustrative purposes only and are not intended to limit the present invention.

EXAMPLES

To generate monoclonal antibodies targeting ROR1 with improved properties, anti-ROR1 antibodies were generated using conventional hybridoma technology. Then, ROR1-mAb004, an antibody that binds to ROR1 at the C-terminus of the Ig-like domain of ROR1, was selected and characterized. The ROR1-mAb004 sequence was further humanized by a conventional CDR grafting method. Humanized sequences were designed. Some of these sequences were expressed as recombinant FIT-Igs and their binding affinity was studied.

We constructed the FIT-Ig protein FIT1007-12B-17, and also produced its MAT-Fab analogue, MAT1007-12B-17, and a reference protein with low affinity for CD3, FIT1007-12B-18. Overall, with identical variable Ig sequences, the FIT-Ig format demonstrated superior tumor cell killing efficiency in vitro and enhanced cytokine release compared to MAT-Fab. Reduced affinity for CD3 also resulted in reduced efficiency of redirected T cell cytotoxicity (RTCC).

Both FIT-Ig and MAT-Fab demonstrated ROR1 target-dependent T cell activation in a co-culture reporter gene assay, indicating a possible inefficiency of T cell activation in the absence of the ROR1 target. This phenomenon is consistent with the difference in CD3 binding activity between FIT-Ig and the parental anti-CD3 mAb.

FIT-Ig and MAT-Fab demonstrated significant in vivo efficacy in a triple-negative breast cancer xenograft model.

Example 1. Obtaining antibodies against ROR1

Antibodies against ROR1 were generated by immunizing Balb/c or SJL Q30-Y406 mice with human ROR1, a recombinant extracellular domain of human ROR1 (UniProt identifier: Q01973-1):

>HUMAN_ROR1_ECD

(SEQ ID NO: 41)

Mice were immunized at 2-week intervals and serum titers were monitored weekly after the second injection. After 4-6 immunizations, splenocytes were collected and fused with mouse myeloma cells to form hybridoma cell lines. Fusion products were plated in hypoxanthine-aminopterin-thymidine (HAT)-containing selective medium in 96-well plates at a density of 1 x 10 ⁵ spleen cells per well. Macroscopic hybridoma colonies were observed seven to ten days after fusion. Hybridoma cell supernatants were then screened and selected to identify cell lines producing mouse ROR1-specific antibodies. After preliminary characterization, one anti-ROR1 antibody, ROR1-mAb004, was selected and sequenced.

Example 1.1 Sequences of variable regions of heavy and light chains

To amplify the variable regions of the heavy and light chains, total RNA of each hybridoma clone was isolated from more than 5 x ¹⁰⁶ cells using TRIzol™ RNA Isolation Reagent (bivitrogen, Cat. #15596018). cDNA was synthesized using the Invitrogen™ Superscript™ III First-Strand Synthesis SuperMix Kit (ThermoFisher Scientific, Cat. #18080) according to the manufacturer's instructions; cDNA encoding the variable regions of the mouse immunoglobulin light and heavy chains was amplified using the MilliporeSigma™ Novagen™ Mouse Ig-Primer Set (Fisher Scientific, Cat. #698313). The PCR products were analyzed by 1.2% agarose gel electrophoresis with SYBR™ Safe DNA Gel Stain (ThermoFisher, Cat.# S33102). DNA fragments of the correct size were purified using the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Cat.# 740609) according to the manufacturer's instructions and subcloned into the pMD18-T vector individually. Fifteen colonies were picked after each transformation and the sequences of the inserted fragments were analyzed by DNA sequencing. The protein sequences of the mouse mAb variable regions were analyzed by homologous sequence alignment.

The variable domain sequence for the selected anti-ROR1 antibody is shown in the table below. Complementarity determining regions (CDRs) are underlined based on Kabat numbering.

Example 1.2 Kinetics of binding of antibodies against ROR1

Binding affinity and kinetic constants of anti-ROR1 antibodies were determined at 25°C using Octet®RED96 Biolayer Interferometry (Pall ForteBio LLC) following standard procedures. Briefly, mouse IgG Fc capture biosensors (AMC) were used to capture purified anti-ROR1 antibodies. The sensors were then immersed in solutions containing recombinant human ROR1-ECD protein to detect binding of the target protein to the captured antibodies. Kinetic constants were determined by processing and fitting the data with a 1:1 binding model using Fortebio analysis software. Table 2 below summarizes the results obtained for ROR1-mAb004 compared to two previously described anti-ROR1 mAbs; ROR1-Tab1 is clone R12 described in WO2014167022, and ROR1-Tab2 is clone D10 described in WO2012097313.

Example 1.3 Characterization of cell surface binding of anti-ROR1 antibodies

The specificity and binding efficiency of anti-ROR1 antibodies were investigated by solid-phase protein ELISA and cell surface binding flow cytometric assay. The EC50 of binding was calculated and the values are shown in Table 3 below. Briefly, the binding properties of anti-ROR1 antibodies were measured by solid-phase ELISA as follows: recombinant ROR1-ECD protein was immobilized at a concentration of 1 μg/ml in 96-well plates at 4°C overnight. The plates were washed once with wash buffer (PBS containing 0.05% Tween 20) and blocked with ELISA blocking buffer (1% BSA in PBS containing 0.05% Tween 20) at room temperature for 2 h. Anti-ROR1 antibodies were then added and incubated at 37°C for 1 h. The plates were washed three times with wash buffer. HRP-labeled anti-mouse IgG secondary antibody (Sigma, Cat. No. A168) was added and the plates were incubated at 37°C for 30 min, then washed 5 times with wash buffer. 100 μl of tetramethylbenzidine (TMB) chromogenic solution were added to each well. After color change, the reaction was stopped with 1 N HCl and the optical density was measured at 450 nm on a Varioskan™ LUX microplate reader (ThermoFisher Scientific). Binding signals were plotted against antibody concentration using GraphPad Prism 6.0 software and EC50 was calculated accordingly. The results are shown in Figure 1. Figure 1 shows the binding activity of ROR1-ECD protein by monoclonal antibodies ROR1-mAb004 and ROR1-Tab1, and non-specific mIgG1 was used as a negative control.

The cell binding activity of anti-ROR1 antibodies was measured using a CHO cell line transfected with human ROR1 (CHO-ROR1) and a ROR1-expressing myeloma cell line (RPMI8226). Briefly, 5 × 10 ⁵ cells were seeded into each well of a 96-well plate. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. Then, 100 μl of serially diluted antibodies were added to each well and mixed with the cells. After 40 min of incubation at 4 °C, the plates were washed several times to remove excess antibodies. Then, a fluorochrome-conjugated goat anti-mouse IgG secondary antibody was added and incubated with the cells at room temperature for 20 min. After another round of centrifugation and a washing step, the cells were resuspended in FACS buffer for reading on a CytoFLEX flow cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readings were plotted versus antibody concentration and analyzed using GraphPad Prism 6.0 software. The results shown in Figures 2A-B illustrate the binding activity of ROR1-mAb004 and ROR1-Tab1 monoclonal antibodies to ROR1-expressing cells. Non-specific mIgG1 was used as a negative control.

Example 1.4. Study of internalization of antibodies against ROR1

Internalization of anti-ROR1 antibodies upon binding was studied using ROR1-expressing myeloma cell line RPMI8226. Cells were picked and resuspended in FACS buffer at a density of 3 million/ml. Diluted antibodies were added to the tubes and incubated for 30 min at 4°C. After the first incubation, the cells were washed three times with cold PBS to remove unbound antibody. Then, the cells of each antibody were divided into two groups for “control” and “internalization”, respectively. Cells in the “internalization” group were resuspended in pre-warmed medium and incubated at 37°C for 2 h to allow internalization, while cells in the “control” group were stored at 4°C for the same period. After the second incubation, the cells were washed once with cold PBS and incubated with fluorescein-labeled secondary antibody for 30 min at 4°C. After another centrifugation cycle and washing step, the cells were resuspended in FACS buffer for reading on a CytoFLEX flow cytometer (Beckman Coulter). A non-specific control mouse IgG was used for background calibration (MFI _background ). The difference between the MFI readings in the “control” and “internalization” groups (ΔMFI) reflects the internalization of anti-ROR1 antibodies, and this difference compared to the calibrated MFI of the “control” reflects the percentage of antibody internalization, which was calculated as follows and is listed in Table 4 below,

Percentage of internalization (ΔMFI) = [1 - (MFI _{internalization} - MFI _background ) / (MFI _control MFI _background ] × 100%

Example 1.5. Binding of antibodies against ROR1 to epitopes.

The binding epitope of anti-ROR1 antibodies was identified by competitive ELISA. Briefly, 1 μg/ml purified antibodies were immobilized on 96-well plates and incubated overnight at 4°C. After washing with PBS containing 0.05% Tween 20, the plates were blocked with blocking buffer (PBS containing 0.05% Tween 20 and 2% BSA) at 37°C for 2 h. Biotinylated human ROR1-ECD protein premixed with anti-ROR1 antibody (sample) or non-specific mouse IgG (baseline) was added to the wells of the plate and incubated at 37°C for 1 h before washing 3 times. Streptavidin-HRP (1:5000 dilution) was then added to each well and incubated at 37°C for 1 hour before washing again 3 times. Chromogenic tetramethylbenzidine (TMB) solution was added for 5 minutes to stain, then the reaction was stopped with 1 M HCl. Absorbance at 450 nm (OD ₄₅₀ ) was measured on a microplate reader. OD450 _intr represents the binding level of human ROR1-ECD to anti-ROR1 antibodies in the absence of competition, while the difference between OD450 _intr and OD450 _sample reflects the competition between anti-ROR1 antibody immobilized on the plate and the antibody in solution. The percentage of inhibition was calculated using the following equation:

% Inhibition = (1 - OD450 _sample / OD450 _original ) × 100%

Table 5 below shows the competitive ELISA results in terms of percent inhibition, indicating that ROR1-mAb004 competes with ROR1-Tab2 but does not compete with ROR1-Tab1.

Example 2. Design of humanization of ROR1-mAb004

The variable region genes of ROR1-mAb004 were used for humanization engineering. In the first step of this process, the amino acid sequences of the VH and VL domains of ROR1-mAb004 were compared with an available human Ig V gene sequence database to find sequences that best matched the human germline Ig V gene overall. In addition, the VH or VL framework region 4 segment was compared with a J region database to find the human framework region that best matched the mouse VH and VL regions, respectively. Gene 018 best matched the human light chain V gene, and gene VH1-69 best matched the human heavy chain V gene. Humanized variable domain sequences were then constructed in which CDR-L1, CDR-L2, and CDR-L3 of the light chain VL domain of ROR1-mAb004 were grafted onto the 018 gene framework sequences with JK4 framework 4 sequence after CDR-L3, respectively; and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain VH domain of ROR1-mAb004 were grafted onto the VH1-69 framework sequences with JH6 framework 4 sequence after CDR-H3. A three-dimensional model of the Fv of ROR1-mAb004 was then generated to determine whether there were any positions in the framework where mouse amino acids were involved in supporting loop structures or the VH/VL interface. To maintain affinity/activity, these residues in the humanized sequences can be back-mutated to mouse antibody-specific residues at the same positions. Several desirable back-mutations were identified in the VH and VL of ROR1-mAb004 and alternative VH and VL arrangements were constructed, as shown in Table 6 below.

In addition, in order to avoid potential asparagine deamidation introduced by two "NG" (Asn-Gly) amino acids in CDR-H2 of ROR1-mAb004, four mouse VH sequences were constructed with different point mutations, which are shown in the last four VH sequences in Table 6. For information on asparagine deamidation induced by "NG" (Asn-Gly) amino acids in CDR-H2 of antibody and its impact on antibody stability, see, e.g., Qingrong Yan et al., (2018) Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies, mAb, 10: 6, 901–912.

Example 3. Production and characterization of humanized anti-CD3 antibody

The hybridoma-derived anti-CD3 monoclonal antibody mAbCD3-001 was generated and selected using conventional hybridoma technology and then humanized using a conventional CDR grafting technique. The humanized VH sequences were then backmutated and an NS mutation was introduced to replace NA in the humanized kappa chain to eliminate the propensity for asparagine deamidation (details are provided in PCT/CN/120991, which is incorporated herein by reference in its entirety). The resulting humanized VH and VL constructs are shown in Table 7 (below).

Pairing of human VH and human VK sequences yielded two humanized antibodies designated HuEM0006-01-24 (with VH/VL pair SEQ ID NOs: 22 and 24) and HuEM0006-01-27 (with VH/VL pair SEQ ID NOs: 23 and 24) (Table 7). The recombinant humanized mAbs were transiently expressed in HEK293 cells and purified by protein A chromatography.

The binding activity of humanized anti-CD3 antibodies was studied by flow cytometry using the Jurkat T cell line expressing human CD3. 5× ¹⁰⁵ Jurkat cells in FACS buffer were seeded into each well of a 96-well plate. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. Then 100 μl of serially diluted antibodies were added to each well and mixed with the cells. After 40 min of incubation at 4°C, the plates were washed several times to remove excess antibodies. A fluorochrome-conjugated secondary antibody (Alexa Fluor® 647, goat anti-human IgG1 H&L; Jackson ImmunoResearch, Cat# 109-606-170) was then added and incubated with the cells at room temperature for 20 min. After another centrifugation cycle and a wash step, the cells were resuspended in FACS buffer for reading on a CytoFLEX flow cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readings were plotted versus antibody concentration and analyzed using GraphPad Prism 5.0 software. The HuEMO006-01-24 antibody showed higher CD3 binding affinity than the HuEM0006-01-27 antibody.

Example 4. Obtaining FIT-Ig against ROR1/CD3

A panel of FIT-Ig proteins recognizing both human ROR1 and human CD3 was constructed using the VH/VL sequences in Table 6 as the anti-ROR1 panel, the VH/VL sequences in Table 7 as the anti-CD3 panel, and the human constant region sequences in Table 8.

FIT-Ig molecules were constructed according to the general procedures described in PCT publication WO 2015/103072. Each FIT-Ig consisted of three polypeptide chains with the following structures:

Chain No. 1 (long chain): VL _A -CL-VH _B -CH1-joint-CH2-CH3;

Chain #2 (first short chain): VH _A -CH1;

Chain #3 (second short chain): VL _B -CL;

where A is ROR1, B is CD3, VL _ROR1 is a light chain variable domain of a humanized monoclonal antibody that recognizes ROR1, VH _CD3 is a heavy chain variable domain of a humanized monoclonal antibody that recognizes CD3, VL _CD3 is a light chain variable domain of a humanized monoclonal antibody that recognizes CD3, VH _ROR1 is a heavy chain variable domain of a humanized monoclonal antibody that recognizes ROR1, each CL is a light chain constant domain (SEQ ID NO: 32), each CH1 is the first heavy chain constant domain (SEQ ID NO: 33), and CH1-hinge-CH2-CH3 is the C-terminal constant region of the heavy chain from CH1 to the end of the Fc region (SEQ ID NO: 31).

To construct the long-strand vector, cDNA encoding the VL _ROR1 -CL-VH _CD3 segment was synthesized de novo and inserted into the multiple cloning site (MCS) of a vector containing the human CH1-hinge-CH2-CH3 coding sequences. The MCS sequence in the resulting vector was removed during homologous recombination to ensure that all domain fragments were in the correct reading frame. Similarly, to construct the first and second short strands, the VH _ROR1 and VL _CD3 structural genes were synthesized de novo and inserted into the MCS of the corresponding vectors containing the coding segments for the human CH1 and CL domains, respectively.

The pairing of humanized VH and humanized VL allowed the generation of humanized ROR1/CD3 binding proteins FIT-Ig listed in Table 9 below. In addition, a chimeric antibody (FIT1007-12B) with the original VH/VL sequences of mouse ROR1-mAb004 and human constant sequences was generated as a positive control for ranking the humanized binding proteins.

The recombinant FIT-Ig proteins listed in Table 10 were produced by transient expression and purified as described herein. Three plasmids for the three polypeptide chains of each FIT-Ig construct, respectively, were co-transfected into HEK 293F cells. After approximately six days of cell culture following transfection, the supernatant was collected and subjected to protein A affinity chromatography. The composition and purity of the purified antibodies were analyzed by size exclusion chromatography (SEC). The purified antibody in PBS was loaded onto a TSKgel SuperSW3000 SEC column, 300 x 4.6 mm (TOSOH). A DIONEX™ UltiMate 3000 HPLC instrument (Thermo Scientific) was used for SEC using UV detection at 280 nm and 214 nm. The expression and SEC-HPLC results are presented below in Table 10.

ROR1/CD3 FIT-Ig proteins were analyzed and ranked by dissociation rate constant (k _off ) using Octet®RED96 biolayer interferometry (Pall FortéBio LLC). Fc hIgG capture biosensors (ANS) (Pall) were first exposed to 100 nM antibody for 30 seconds to capture the antibody, then immersed in running buffer (1X pH 7.2 PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to check the baseline. The sensors with captured antibody were immersed in 10 μg/mL recombinant human ROR1 ECD protein for 5 minutes to measure association, then immersed in running buffer for 1200 seconds to measure dissociation. The association and dissociation curves were fitted with a 1:1 Langmuir binding model using FortéBio data analysis software (Pall). The results are shown in Table 10 below. Dissociation rate ratios were calculated from the antibody dissociation rate relative to the FIT 1007-12B dissociation rate. A lower ratio indicates slower dissociation of the antibody compared to the parent chimeric FIT 1007-12B antibody.

The VH/VL humanization design of FIT1007-12B-1 was selected based on the highest binding activity. In addition, the CDR-H2 point mutation design of FIT 1007-12B-13 showed higher expression titer and binding activity compared with the other design. The mutation design "ROR1-mAb004VH(AA)" (SEQ ID NO: 17) was selected to be combined with the VH humanization design "ROR1-mAb004VH.1a" (SEQ ID NO: 10) to generate candidate molecules. The humanized VH sequence, namely ROR1-mAb004VH.1a(AA), is shown below:

>ROR1-mAb004VH.la(AA) (SEQ ID NO: 21)

Example 5. Construction and expression of ROR1/CD3 FIT-Ig and MAT-Fab

The method shown in Example 4 was used to construct FIT-Ig. No linkers were used between the immunoglobulin domains. The complete sequences of the FIT-Ig binding proteins are provided in the sequence information in Table 11.

In addition, a panel of ROR1/CD3 MAT-Fab proteins with the same VH/VL sequence combination was constructed according to the procedure described in WO 2018/035084. Each MAT-Fab consisted of four polypeptide chains with the following structures:

Chain No. 1 (long chain with a “protrusion”): VL _A -CL-VH _B -CH1-joint-CH2-CH3;

Chain #2 (first short chain): VH _A -CH1;

Chain #3 (second short chain): VL _B -CL;

Chain No. 4 (Fc "hollow"): hinge-CH2-CH3;

where chain #1 comprises a mutant human constant IgG1 with the mutation S354C, T366W as a “knob”, chain #4 is an Fc chain with the mutation Y349C, T366S, L368A, Y407V as a “hole”, where A is ROR1 and B is CD3.

By a cloning method similar to that shown previously for FIT-Ig, the VH/VL genes of the MAT-Fab polypeptide chains were synthetically produced and then cloned into vectors containing the corresponding constant domains, respectively. The complete sequences of the MAT-Fab proteins are presented in the sequence information in Table 12.

Recombinant FIT-Ig and MAT-Fab proteins were produced by transient expression and purified as described herein. Three or four plasmids encoding the corresponding polypeptide chains of each FIT-Ig or MAT-Fab, respectively, were co-transfected into HEK 293F cells. After approximately six days of cell culture following transfection, the supernatant was collected and subjected to protein A affinity chromatography. The composition and purity of the purified antibodies were analyzed by size exclusion chromatography (SEC). The purified antibody in PBS was loaded onto a TSKgel SuperSW3000 SEC column, 300 x 4.6 mm (TOSOH). A DIONEX™ UltiMate 3000 HPLC instrument (Thermo Scientific) was used for SEC using UV detection at 280 nm and 214 nm. The expression and SEC-HPLC results are shown below in Table 13.

The ROR1 binding affinity/kinetics of the humanized candidate FIT1007-12B-17 and the parental chimeric FIT-Ig FIT1007-12B were measured using the method described in Example 3. For each antibody, the measurements were titrated with 6 antigen concentrations, i.e., at 3-fold dilutions, starting at 500 nM. The binding kinetics and affinities are shown in Table 14 below. The binding kinetics of FIT1007-12B-18, MAT1007-12B-17, and MAT1007-12B-18 are similar to those of FIT1007-12B-17. These candidates contain the same ROR1-binding Fab.

Example 6. Study of the binding characteristics of humanized FIT-Ig and MAT-Fab

The binding activity of ROR1×CD3 antibodies to cells was measured using a CHO cell line transfected with the human TCR/CD3 complex (CHO-CD3-TCR) and tumor cell lines expressing ROR1 (NCI-H1975, MDA-MB-231, A549, and RPMI8226). Briefly, 5× ¹⁰⁵ cells were seeded into each well of a 96-well plate. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. Then, 100 μl of serially diluted antibodies were added to each well and mixed with the cells. After 40 min of incubation at 4°C, the plates were washed several times to remove excess antibodies. A fluorochrome-conjugated goat anti-human IgG secondary antibody was then added and incubated with the cells at room temperature for 20 min. After another centrifugation cycle and a washing step, the cells were resuspended in FACS buffer for reading on a CytoFLEX flow cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readings were plotted against antibody concentration and analyzed using GraphPad Prism 6.0 software.

As shown in Figure 3, the binding activity of CHO-CD3-TCR correlated with the binding affinity of CD3 and the valence of each molecule. When FIT-Ig was compared with the parental anti-CD3 monoclonal IgG1 antibody, i.e., FIT1007-12B-17 with HuEM0006-01-24 (VH/VL sequences: SEQ ID NOs: 22 and 24, Table 7) or FIT1007-12B-18 with HuEM0006-01-27 (VH/VL sequences: SEQ ID NOs: 23 and 24, Table 7), FIT-Ig showed a relatively lower binding efficiency, which may be due to steric hindrance.

As shown in Figure 4A-D, the binding activity to ROR1-expressing tumor cells is relatively similar between FIT-Ig and their common parental anti-ROR1 monoclonal antibody (HuROR1-mAb004-1, with sequences of ROR1-mAb004VH.1a (AA) and ROR1-mAb004VK.1a, SEQ ID NOs: 21 and 13). The binding curve of MAT-Fab is different from that of FIT-Ig and the parental anti-ROR1 monoclonal antibody, which may be due to the different binding valency to the target.

Example 7. Redirected CD3 activation by humanized FIT-Ig and MAT-Fab

A co-culture reporter gene assay was used to measure redirected CD3 activation by the ROR1×CD3 bispecific FIT-Ig and MAT-Fab antibodies. In this assay, Jurkat-NFAT-luc cells activate a downstream luciferase signal upon activation of cell surface CD3. RPMI8226 cells were used as an ROR1-expressing target cell capable of cross-linking the CD3/TCR complex on T cells via the ROR1×CD3 bispecific antibody upon binding of ROR1. Jurkat-NFAT-luc and RPMI8226 cells were washed and resuspended in assay medium (RPMI1640 with 10% FBS) separately. Both cell types were seeded in 96-well plates (Costar #3903) at 1× ¹⁰⁵ cells/well at a 1:1 ratio. FIT-Ig or MAT-Fab antibodies were added, mixed with the cells, and incubated for 4 h at 37°C. After incubation, the ONE-Glo™ Luminescence Assay Kit (Promega, Cat. No. E6130) was prepared and added to the wells according to the manufacturer's instructions. The plates were read for luminescent signals using a Varioskan™ LUX Microplate Reader (ThermoFisher Scientific). The results are shown in Figure 5.

In addition, one non-specific negative control FIT-Ig, a bispecific molecule based on anti-EGFR×cMET antibodies (EMB01) and two monoclonal anti-CD3 antibodies, namely HuEM0006-01-24 and HuEM0006-01-27, were tested. All bispecific ROR1×CD3 binding proteins resulted in enhanced T cell activation in the presence of ROR1-expressing target cells compared to monospecific anti-CD3 binding proteins lacking binding activity against ROR1.

Non-specific redirected activation of CD3 was tested using a Jurkat-NFAT-luc based reporter gene assay in the absence of target cells. The results are shown in Figure 6. This assay was performed in the absence of cells expressing the co-target of the bispecific binding proteins, in this case ROR1. The ROR1xCD3 bispecific antibodies demonstrated less non-specific redirected activation than the CD3 antibody alone in the absence of ROR1 expressing target cells.

Example 8. Redirected T-cell cytotoxicity of humanized FIT-Ig and MAT-Fab

The tumor cell killing efficiency of bispecific ROR1×CD3 binding proteins was measured in a T cell redirected cytotoxicity assay using the human breast cancer cell line MDA-MB-231 as target cells and human T cells as effector cells. Briefly, cells were harvested, washed, and resuspended in assay medium (RPMI1640 with 10% FBS). MDA-MB-231 cells were seeded in 96-well flat-bottom plates (Corning, Cat. #3599) at 5 × 10 ⁴ cells/well. T cells were purified from human PBMCs using a commercial PBMC isolation kit (EasySep™, Stemcell Technologies, Cat. #17951) and added to the wells at 2 × 10 ⁵ cells/well. Test antibodies were added and incubated with the cell mixture for 48 hours at 37°C. Lactate dehydrogenase (LDH) release was measured using the CytoTox 96® Cytotoxicity Assay Kit (Promega, Cat.# G1780). OD490 readings were obtained according to the manufacturer's instructions. In addition, maximum and minimum lysis were assessed according to the CytoTox kit instruction manual (Promega, Cat.# G1780). Maximum lysis was obtained by adding lysis buffer to tumor cell-only samples. Minimum lysis was obtained from background culture medium. Minimum lysis was subtracted from all sample readings. Maximum lysis of MDA-MB-231 target cells (100%) minus minimum lysis (0%) was used as the denominator for normalization. Percentage LDH release was plotted against bispecific antibody concentration. As shown in Figure 7, bispecific ROR1×CD3-binding proteins exhibited T cell redirected cytotoxicity against MDA-MB-231 tumor cells, while bispecific EGFR×cMET-binding FIT-Ig EMB01 exhibited low cytotoxic activity.

Example 9. MDA-MB-231 tumor volume in human PBMC-transplanted M-NSG mice treated with ROR1×CD3 bispecific antibodies

Antitumor efficacy was assessed in M-NSG mice, an immunodeficient strain lacking T cells, B cells, and natural killer cells. MDA-MB-231 cells (5×10 ⁶ ) were injected subcutaneously into the right dorsal lateral region. Five days after tumor cell inoculation, mice received a single intraperitoneal dose of 3.5×10 ⁶ human PBMC. Animals were randomized based on tumor size (~150-300 mm ³ ) on day 15 and treatment was started the following day. Tumor growth was monitored by caliper measurements. The study was terminated on day 16 after the first administration, and mice were sacrificed when signs of GVHD appeared. Mice were treated once a week for 3 weeks (1 time/week × 3) with 1 mg/kg PT1007-12B-17, FIT1007-12B-18, MAT 1007-12B-17 or vehicle by intraperitoneal (i.p.) injection. As shown in Figure 8, mice in the experimental group treated with FIT-Ig and MAT-Fab demonstrated significant inhibition of tumor growth compared with the vehicle group (****P<0.0001; compared with the vehicle group, two-way ANOVA combined with Dunnett's test).

Example 10. Study of internalization characteristics of humanized anti-ROR1 antibodies

Internalization of humanized anti-ROR1 antibodies upon binding was studied using ROR1-expressing myeloma cell line RPMI8226 in a manner similar to that described previously in Example 1.4. Briefly, cells were harvested and resuspended in FACS buffer at a density of 3 million cells/ml. Diluted antibodies were added to tubes and incubated for 30 min at 4°C. After the first incubation, cells were washed three times with cold PBS to remove unbound antibody. Cells treated with each antibody were then divided into three groups: 4°C, 37°C, and 37°C + PAO, respectively. The cells in the 37°C internalization group were resuspended in pre-warmed medium and incubated at 37°C for 2 h to allow internalization, while the cells in the 4°C control group were stored at 4°C for the same period. The cells in the 37°C + PAO group were resuspended in pre-warmed medium and incubated in the presence of 3 μM phenylarsine oxide (an endocytosis inhibitor to prevent internalization of membrane proteins) at 37°C for 2 h. The 37°C + PAO experimental group served to calibrate the antibody dissociation effect. After the second incubation, the cells were washed once with cold PBS and incubated with fluorescein-labeled secondary antibody for 30 min at 4°C. After another round of centrifugation and a washing step, the cells were resuspended in FACS buffer for reading on a CytoFLEX flow cytometer (Beckman Coulter). The reading for non-specific control mouse IgG (MFI _background ) was calculated and used for background calibration. The difference between the MFI readings in the “control” and “internalization” groups (ΔMFI) reflects the internalization of anti-ROR1 antibodies, and this difference compared to the calibrated MFI of the “control” reflects the percentage of antibody internalization, which was calculated as follows and is listed in Table 15 below. As shown in Figure 9, at 100 nM concentration, the HuROR1-mAb004-l antibody and its corresponding FIT-Ig/MAT-Fab showed limited internalization. The calculated percentage internalization values of HuROR-mAb004-1 and FIT1007-12B-17 antibodies are consistent with the results shown in Example 1.4, Table 4.

MAT-Fab showed decreased binding at 37°C, which may be due to its lower binding valence and higher binding rate at 37°C. The binding curve for calculating MAT-Fab internalization did not reach a binding plateau at 100 nM.

Percentage of internalization (ΔMFI) = [1 - (MFI _{internalization} - MFI _background ) / (MFI _control - MFI _background )] × 100%

Example 11. Preparation of reference FIT-Ig and comparison of in vitro activity with FIT1007-12B-17

The anti-CD3 antibody sequences shown in Table 7 were used to generate FIT-Ig with the VH/VL sequences of one of two anti-ROR1 reference antibodies, ROR1-Tab1 (clone R12) and ROR1-Tab2 (clone D10). The construction and generation of the reference FIT-Ig was performed as described in Example 3. No linkers were used between the immunoglobulin domains. The complete sequences of the FIT-Ig binding proteins are provided in the sequence information in Tables 16 and 17. The cell surface binding activity of the reference FIT-Ig was assessed using the method described in Example 1.3, and the redirected cytotoxicity activity was assessed using the method described in Example 6.

The VH/VL sequences of one of the two ROR1 reference antibodies, ROR1-Tab1 (clone R12) and ROR1-Tab2 (clone D10), used in this Example are shown below: VH sequence of antibody D10 (SEQ ID NO: 42)

VL sequence of antibody D10 (SEQ ID NO: 43)

VH sequence of antibody R12 (SEQ ID NO: 44)

VL sequence of antibody R12 (SEQ ID NO: 45)

Figure 11 shows a comparison of FIT1007-12B-17 with the reference FIT-Ig molecules presented in Table 16. Figures 11A and 11B illustrate FIT1007-12B-17 and the reference FIT-Ig molecules exhibiting similar binding to the surface of both ROR1-expressing MDA-MB-231 cells and CD3-expressing Jurkat cells. However, as shown in Figure 11C for T cell redirected cytotoxicity against MDA-MB-231 cells, FIT1007-12B-17 achieved greater cytotoxicity than the reference FIT-Ig molecules.

--->

SEQUENCE LIST

<110> EpimAb Biotherapeutics (HK) Limited

<120> ANTIBODIES AGAINST ROR1 AND RELATED BISPECIFIC BINDING PROTEINS

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

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Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

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Lys Val Glu Pro Lys Ser Cys

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

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Ile Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ser Leu Leu Trp

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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

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Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

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Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

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Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

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Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

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Phe Asn Arg Gly Glu Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu

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Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

225 230 235 240

Phe Ser Phe Thr Asn Tyr Tyr Val His Trp Met Arg Gln Ala Pro Gly

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Gln Gly Leu Glu Trp Met Gly Trp Ile Ser Pro Gly Ser Asp Asn Thr

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Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr

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Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

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Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Gly Asn Tyr Tyr Phe

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Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

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Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

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Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

355 360 365

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

370 375 380

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

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Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

405 410 415

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

420 425 430

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

435 440 445

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

450 455 460

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

465 470 475 480

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

485 490 495

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

500 505 510

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

515 520 525

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

530 535 540

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

545 550 555 560

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

565 570 575

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

580 585 590

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

595 600 605

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

610 615 620

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

625 630 635 640

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

645 650 655

Leu Ser Leu Ser Pro Gly Lys

660

<210> 35

<211> 223

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 35

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Tyr Pro Gly Asn Ala Asp Ile Lys Tyr Asn Ala Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala His Ile Tyr Tyr Asp Phe Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 36

<211> 219

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 36

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ala

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 37

<211> 663

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ser Leu Leu Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu

210 215 220

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

225 230 235 240

Phe Ser Phe Thr Asn Tyr Tyr Val His Trp Met Arg Gln Ala Pro Gly

245 250 255

Gln Gly Leu Glu Trp Ile Gly Trp Ile Ser Pro Gly Ser Asp Asn Thr

260 265 270

Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Thr

275 280 285

Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

290 295 300

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Gly Asn Tyr Tyr Phe

305 310 315 320

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

325 330 335

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

340 345 350

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

355 360 365

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

370 375 380

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

385 390 395 400

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

405 410 415

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

420 425 430

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

435 440 445

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

450 455 460

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

465 470 475 480

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

485 490 495

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

500 505 510

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

515 520 525

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

530 535 540

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

545 550 555 560

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

565 570 575

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

580 585 590

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

595 600 605

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

610 615 620

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

625 630 635 640

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

645 650 655

Leu Ser Leu Ser Pro Gly Lys

660

<210> 38

<211> 663

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ser Leu Leu Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu

210 215 220

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

225 230 235 240

Phe Ser Phe Thr Asn Tyr Tyr Val His Trp Met Arg Gln Ala Pro Gly

245 250 255

Gln Gly Leu Glu Trp Met Gly Trp Ile Ser Pro Gly Ser Asp Asn Thr

260 265 270

Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr

275 280 285

Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

290 295 300

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Gly Asn Tyr Tyr Phe

305 310 315 320

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

325 330 335

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

340 345 350

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

355 360 365

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

370 375 380

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

385 390 395 400

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

405 410 415

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

420 425 430

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

435 440 445

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

450 455 460

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

465 470 475 480

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

485 490 495

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

500 505 510

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

515 520 525

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

530 535 540

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

545 550 555 560

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

565 570 575

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

580 585 590

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

595 600 605

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

610 615 620

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

625 630 635 640

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

645 650 655

Leu Ser Leu Ser Pro Gly Lys

660

<210> 39

<211> 231

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 39

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1 5 10 15

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

130 135 140

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 40

<211> 663

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ser Leu Leu Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu

210 215 220

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

225 230 235 240

Phe Ser Phe Thr Asn Tyr Tyr Val His Trp Met Arg Gln Ala Pro Gly

245 250 255

Gln Gly Leu Glu Trp Ile Gly Trp Ile Ser Pro Gly Ser Asp Asn Thr

260 265 270

Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Thr

275 280 285

Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

290 295 300

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Gly Asn Tyr Tyr Phe

305 310 315 320

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

325 330 335

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

340 345 350

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

355 360 365

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

370 375 380

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

385 390 395 400

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

405 410 415

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

420 425 430

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

435 440 445

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

450 455 460

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

465 470 475 480

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

485 490 495

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

500 505 510

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

515 520 525

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

530 535 540

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

545 550 555 560

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

565 570 575

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

580 585 590

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

595 600 605

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

610 615 620

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

625 630 635 640

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

645 650 655

Leu Ser Leu Ser Pro Gly Lys

660

<210> 41

<211> 377

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 41

Gln Glu Thr Glu Leu Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser

1 5 10 15

Trp Asn Ile Ser Ser Glu Leu Asn Lys Asp Ser Tyr Leu Thr Leu Asp

20 25 30

Glu Pro Met Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr Ala Glu Leu

35 40 45

His Cys Lys Val Ser Gly Asn Pro Pro Pro Thr Ile Arg Trp Phe Lys

50 55 60

Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Leu Ser Phe Arg Ser

65 70 75 80

Thr Ile Tyr Gly Ser Arg Leu Arg Ile Arg Asn Leu Asp Thr Thr Asp

85 90 95

Thr Gly Tyr Phe Gln Cys Val Ala Thr Asn Gly Lys Glu Val Val Ser

100 105 110

Ser Thr Gly Val Leu Phe Val Lys Phe Gly Pro Pro Pro Thr Ala Ser

115 120 125

Pro Gly Tyr Ser Asp Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro Tyr

130 135 140

Arg Gly Ile Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met

145 150 155 160

Glu Ser Leu His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala

165 170 175

Phe Thr Met Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln

180 185 190

Phe Ala Ile Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys Asp Glu

195 200 205

Thr Ser Ser Val Pro Lys Pro Arg Asp Leu Cys Arg Asp Glu Cys Glu

210 215 220

Ile Leu Glu Asn Val Leu Cys Gln Thr Glu Tyr Ile Phe Ala Arg Ser

225 230 235 240

Asn Pro Met Ile Leu Met Arg Leu Lys Leu Pro Asn Cys Glu Asp Leu

245 250 255

Pro Gln Pro Glu Ser Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile

260 265 270

Pro Met Ala Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn Ser Thr

275 280 285

Gly Val Asp Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg Gln

290 295 300

Cys Gln Pro Trp Asn Ser Gln Tyr Pro His Thr His Thr Phe Thr Ala

305 310 315 320

Leu Arg Phe Pro Glu Leu Asn Gly Gly His Ser Tyr Cys Arg Asn Pro

325 330 335

Gly Asn Gln Lys Glu Ala Pro Trp Cys Phe Thr Leu Asp Glu Asn Phe

340 345 350

Lys Ser Asp Leu Cys Asp Ile Pro Ala Cys Asp Ser Lys Asp Ser Lys

355 360 365

Glu Lys Asn Lys Met Glu Ile Leu Tyr

370 375

<210> 42

<211> 117

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 42

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Phe Thr Asn Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Leu Leu

65 70 75 80

Lys Met Thr Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Arg Gly Ser Ser Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 43

<211> 106

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 43

Glu Ile Val Leu Ser Gln Ser Pro Ala Ile Thr Ala Ala Ser Leu Gly

1 5 10 15

Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Asn Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Arg Ser Gly Thr Ser Pro Arg Pro Trp Ile Tyr

35 40 45

Glu Ile Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Asn Tyr Pro Leu Ile Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Gln

100 105

<210> 44

<211> 121

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 44

Gln Glu Gln Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Ser Ala Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr Tyr Ala Thr Trp Val

50 55 60

Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Gln Asn Thr Val Asp

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Ala Ala Asp Arg Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala Leu Phe Asn Ile Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Ile Ser Ser

115 120

<210> 45

<211> 112

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 45

Glu Leu Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser

1 5 10 15

Pro Ala Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr

20 25 30

Ile Asp Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met

35 40 45

Gln Val Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro

65 70 75 80

Ser Val Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr

85 90 95

Ile Gly Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly

100 105 110

<210> 46

<211> 662

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 46

Glu Ile Val Leu Ser Gln Ser Pro Ala Ile Thr Ala Ala Ser Leu Gly

1 5 10 15

Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Asn Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Arg Ser Gly Thr Ser Pro Arg Pro Trp Ile Tyr

35 40 45

Glu Ile Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Asn Tyr Pro Leu Ile Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Gln Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

210 215 220

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe

225 230 235 240

Ser Phe Thr Asn Tyr Tyr Val His Trp Met Arg Gln Ala Pro Gly Gln

245 250 255

Gly Leu Glu Trp Met Gly Trp Ile Ser Pro Gly Ser Asp Asn Thr Lys

260 265 270

Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser

275 280 285

Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr

290 295 300

Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Gly Asn Tyr Tyr Phe Asp

305 310 315 320

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

325 330 335

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

340 345 350

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

355 360 365

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

370 375 380

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

385 390 395 400

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

405 410 415

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

420 425 430

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

435 440 445

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

450 455 460

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

465 470 475 480

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

485 490 495

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

500 505 510

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

515 520 525

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

530 535 540

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

545 550 555 560

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

565 570 575

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

580 585 590

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

595 600 605

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

610 615 620

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

625 630 635 640

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

645 650 655

Ser Leu Ser Pro Gly Lys

660

<210> 47

<211> 220

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 47

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Phe Thr Asn Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Leu Leu

65 70 75 80

Lys Met Thr Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Arg Gly Ser Ser Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 48

<211> 219

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 48

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ala

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 49

<211> 667

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 49

Glu Leu Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser

1 5 10 15

Pro Ala Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr

20 25 30

Ile Asp Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met

35 40 45

Gln Val Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro

65 70 75 80

Ser Val Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr

85 90 95

Ile Gly Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly

100 105 110

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

115 120 125

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

130 135 140

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

145 150 155 160

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

165 170 175

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

180 185 190

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

195 200 205

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Glu Val Gln Leu Val Gln

210 215 220

Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys

225 230 235 240

Lys Ala Ser Gly Phe Ser Phe Thr Asn Tyr Tyr Val His Trp Met Arg

245 250 255

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Ser Pro Gly

260 265 270

Ser Asp Asn Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met

275 280 285

Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu

290 295 300

Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Gly

305 310 315 320

Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

325 330 335

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

340 345 350

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

355 360 365

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

370 375 380

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

385 390 395 400

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

405 410 415

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

420 425 430

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

435 440 445

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

450 455 460

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

465 470 475 480

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

485 490 495

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

500 505 510

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

515 520 525

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

530 535 540

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

545 550 555 560

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

565 570 575

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

580 585 590

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

595 600 605

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

610 615 620

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

625 630 635 640

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

645 650 655

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 50

<211> 224

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 50

Gln Glu Gln Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Ser Ala Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr Tyr Ala Thr Trp Val

50 55 60

Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Gln Asn Thr Val Asp

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Ala Ala Asp Arg Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala Leu Phe Asn Ile Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Ile Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 51

<211> 219

<212> PROTEIN

<213> artificial sequence

<220>

<223> artificial construct

<400> 51

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ala

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (84)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to the orphan receptor tyrosine kinase-like 1 (ROR1) and comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-H1 contains the sequence RSWMN (SEQ ID NO: 1); CDR-H2 contains the sequence RIYPGNGDIKYNGNFKG (SEQ ID NO: 2) or RIYPGNADIKYNANFKG (SEQ ID NO: 4); CDR-H3 contains the sequence IYYDFYYALDY (SEQ ID NO: 3); CDR-L1 contains the sequence KASQDINKYIT (SEQ ID NO: 5); CDR-L2 contains the sequence YTSTLQP (SEQ ID NO: 6); and CDR-L3 contains the sequence LQYDSLLWT (SEQ ID NO: 7), wherein the said CDRs are defined in accordance with the Kabat numbering. 2. An isolated antibody or antigen-binding fragment according to claim 1, characterized in that said antibody comprises a heavy chain variable domain VH and a light chain variable domain VL, wherein: said VH domain comprises a sequence selected from any of SEQ ID NOs: 10-12 and 21, and/or said VL domain comprises a sequence selected from any of SEQ ID NOs: 13-16; or said VH domain comprises the sequence of SEQ ID NO:8 or 17, and/or said VL domain comprises the sequence of SEQ ID NO:9. 3. An isolated antibody or antigen-binding fragment according to claim 1, characterized in that said antibody is a chimeric or humanized antibody, wherein optionally said antibody is a humanized antibody, and wherein the VH domain of said antibody comprises amino acid residues 1E, 27Y and 94H and from 0 to 4 residues selected from 38K, 48I, 66K and 67A, according to Kabat numbering; and said VL domain comprises amino acid residue 71Y and from 0 to 4 residues selected from 4L, 49H, 58I and 69R, according to Kabat numbering. 4. An isolated antibody or antigen-binding fragment according to claim 1, characterized in that said antibody comprises a combination of VH and VL sequences selected from the group consisting of: combinations VH sequence VL sequence 15 SEQ ID NO:21 SEQ ID NO:13 16 SEQ ID NO:21 SEQ ID NO:14 17 SEQ ID NO:21 SEQ ID NO:15 18 SEQ ID NO:21 SEQ ID NO:16 1 SEQ ID NO:8 SEQ ID NO:9 2 SEQ ID NO:17 SEQ ID NO:9 3 SEQ ID NO:10 SEQ ID NO:13 4 SEQ ID NO:10 SEQ ID NO:14 5 SEQ ID NO:10 SEQ ID NO:15 6 SEQ ID NO:10 SEQ ID NO:16 7 SEQ ID NO: 11 SEQ ID NO:13 8 SEQ ID NO: 11 SEQ ID NO:14 9 SEQ ID NO: 11 SEQ ID NO:15 10 SEQ ID NO: 11 SEQ ID NO:16 11 SEQ ID NO:12 SEQ ID NO:13 12 SEQ ID NO:12 SEQ ID NO:14 13 SEQ ID NO:12 SEQ ID NO:15 14 SEQ ID NO:12 SEQ ID NO:16 said antibody optionally comprises a VH domain comprising the sequence of SEQ ID NO: 21 and a VL domain comprising the sequence of SEQ ID NO: 13. 5. An isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 1-4, characterized in that said antibody has one or more of the following characteristics: (i) when bound to the surface of ROR1-expressing cells, said antibody is internalized by no more than 20%, optionally no more than 15% or 14%, 13%, 12%, 11%, as measured in a cell-based assay, wherein internalization may be reflected by a decrease in the percentage of median fluorescence intensity (MFI) detected by flow cytometry when said antibody is bound to the surface of ROR1-expressing cells after a two-hour incubation at 37°C compared to a control maintained at 4°C for the same period; (ii) said antibody binds to human ROR1 at the C-terminus of the Ig-like domain of ROR1 and optionally competes with an antibody having the VH/VL sequence pair of SEQ ID NOs: 42 and 43 for binding to ROR1; (iii) binding of the said antibody to ROR1 induces antitumor activity. 6. A conjugate for specific binding to ROR1, comprising an isolated antibody or antigen-binding fragment according to any one of claims 1-5, conjugated to a drug or to a detection agent. 7. A method for detecting ROR1 in a biological sample, comprising: bringing said biological sample into contact with an isolated antibody or antigen-binding fragment according to any one of claims 1-5 or a conjugate according to claim 6, and determining whether binding to a target antigen is present or absent, thereby inferring the presence or absence of the target in a biological sample. 8. A bispecific binding protein that specifically binds ROR1 and CD3, containing: a) the first antigen-binding site that specifically binds ROR1; and b) a second antigen-binding site that specifically binds CD3, wherein said first antigen-binding site comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, wherein: CDR-H1 contains the sequence RSWMN (SEQ ID NO:1), CDR-H2 contains the sequence RIYPGNGDIKYNGNFKG (SEQ ID NO: 2) or RIYPGNADIKYNANFKG (SEQ ID NO: 4), CDR-H3 contains the sequence IYYDFYYALDY (SEQ ID NO: 3), CDR-L1 contains the sequence KASQDINKYIT (SEQ ID NO: 5), CDR-L2 contains the sequence YTSTLQP (SEQ ID NO: 6), and CDR-L3 contains the sequence LQYDSLLWT (SEQ ID NO: 7), wherein the said CDRs are defined in accordance with the Kabat numbering. 9. The bispecific binding protein of claim 8, wherein said first antigen-binding site comprises a VH domain and a VL domain as defined in any one of claims 2-4. 10. A bispecific binding protein according to claim 8 or 9, characterized in that said second antigen-binding site comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, wherein: CDR-H1 contains the sequence NYYVH (SEQ ID NO:25); CDR-H2 contains the sequence WISPGSDNTKYNEKFKG (SEQ ID NO: 26); CDR-H3 contains the sequence DDYGNYYFDY (SEQ ID NO: 27); CDR-L1 contains the sequence KSSQSLLNARTRKNYLA (SEQ ID NO: 28); CDR-L2 contains the WASTRES sequence (SEQ ID NO: 29); and CDR-L3 contains the sequence KQSYILRT (SEQ ID NO: 30), wherein the said CDRs are defined in accordance with the Kabat numbering. 11. The bispecific binding protein according to claim 10, characterized in that said second antigen-binding site contains: a VH domain comprising the sequence of SEQ ID NO: 22 or 23, and/or VL domain containing the sequence SEQ ID NO: 24. 12. A bispecific binding protein according to any one of claims 8-11, comprising a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, moreover (i) said first polypeptide chain comprises from N-terminus to C-terminus VL _A -CL-VH _B -CH1-Fc, wherein CL is fused directly to VH _B ; said second polypeptide chain comprises from N-terminus to C-terminus VH _A -CH1; said third polypeptide chain comprises from N-terminus to C-terminus VL _B -CL; or (ii) said first polypeptide chain comprises from N-terminus to C-terminus VH _A -CH1-VL _B -CL-Fc, wherein CH1 is fused directly to VL _B ; said second polypeptide chain comprises from N-terminus to C-terminus VL _A -CL; said third polypeptide chain comprises from N-terminus to C-terminus VH _B -CH1; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region optionally comprising from N-terminus to C-terminus a hinge-CH2-CH3, wherein VL _A -CL pairs with VH _A -CH1 to form a first Fab that specifically binds a first antigen A, and VL _B -CL pairs with VH _B -CH1 to form a second Fab that specifically binds a second antigen B, and wherein said first antigen A is ROR1 and said second antigen B is CD3, wherein two of said first polypeptide chains, two of said second polypeptide chains and two of said third polypeptide chains associate to form a FIT-Ig protein. 13. The bispecific binding protein according to claim 12, characterized in that: said first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34 or 37, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35, and said third polypeptide chain comprises the amino acid sequence SEQ ID NO: 36. 14. A bispecific binding protein according to any one of claims 8-11, comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain, moreover (i) said first polypeptide chain comprises, from N-terminus to C-terminus, VL _A -CL-VH _B -CH1-Fc, wherein CL is fused directly to VH _B ; said second polypeptide chain comprises, from N-terminus to C-terminus, VH _A -CH1; said third polypeptide chain comprises, from N-terminus to C-terminus, VL _B -CL; said fourth polypeptide chain comprises Fc; or (ii) said first polypeptide chain comprises from N-terminus to C-terminus VH _A -CH1-VL _B -CL-Fc, wherein CH1 is fused directly to VL _B ; said second polypeptide chain comprises from N-terminus to C-terminus VL _A -CL; said third polypeptide chain comprises from N-terminus to C-terminus VH _B -CH1; said fourth polypeptide chain comprises Fc; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region optionally comprising from N-terminus to C-terminus a hinge-CH2-CH3, wherein VL _A -CL pairs with VH _A -CH1 to form a first Fab that specifically binds a first antigen A, and VL _B -CL pairs with VH _B -CH1 to form a second Fab that specifically binds a second antigen B, and wherein said first antigen A is ROR1 and said second antigen B is CD3, wherein said first polypeptide chain, said second polypeptide chain, said third polypeptide chain and said fourth polypeptide chain associate to form a MAT-Fab protein, wherein the Fc of said first polypeptide chain and the Fc of said fourth polypeptide chain optionally comprise heterodimerizing modifications, especially in the CH3 domain, that facilitate heterodimerization rather than homodimerization of said two chains, additionally, said first polypeptide chain optionally comprises a human IgG1 Fc region with the T366W mutation as a "knob", and said fourth polypeptide chain comprises a human IgG1 Fc region with the T366S, L368A and Y407V mutations as a "hole"; and/or said first polypeptide chain comprises a human IgG1 Fc region with S354C, and said fourth polypeptide chain comprises a human IgG1 Fc region with the Y349C mutation to produce an additional disulfide bridge in the CH3 domain. 15. The bispecific binding protein according to claim 14, characterized in that: said first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38 or 40, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36; and said fourth polypeptide chain comprises the amino acid sequence SEQ ID NO: 39. 16. A nucleic acid molecule encoding a bispecific binding protein according to any one of claims 8-15. 17. A host cell for expressing a bispecific binding protein that specifically binds ROR1 and CD3, comprising the nucleic acid molecule of claim 16. 18. A pharmaceutical composition for treating a disorder in which ROR1 activity is detrimental, comprising an isolated antibody or antigen-binding fragment according to any one of claims 1-5, a conjugate according to claim 6, a bispecific binding protein according to any one of claims 8-15, a nucleic acid according to claim 16 or a host cell according to claim 17, and a pharmaceutically acceptable carrier. 19. Use of an isolated antibody or antigen-binding fragment according to any one of claims 1 to 5, a conjugate according to claim 6, or a bispecific binding protein according to any one of claims 8 to 15 for the manufacture of a medicament for the treatment of a disorder in which ROR1 activity is detrimental. 20. A method for treating a disorder in which ROR1 activity is detrimental, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 18. 21. The use according to paragraph 19 or the method according to paragraph 20, characterized in that the said subject is a human being. 22. The use according to claim 19 or the method according to claim 20, characterized in that said disorder is cancer. 23. The use according to claim 19 or the method according to claim 20, characterized in that said disorder is ROR1-positive hematological malignancies or ROR1-positive solid tumor. 24. The use according to claim 19 or the method according to claim 20, characterized in that said disorder is chronic lymphocytic leukemia (CLL), lung cancer, breast cancer or triple-negative breast cancer.