TW202305000A - Oncolytic viruses expressing anti-ror1/anti-cd3 bispecific antibodies - Google Patents

Abstract

The present disclosure provides bispecific antibodies that bind to ROR1 and CD3 and oncolytic viruses encoding nucleic acid construct encoding such bispecific antibodies. Also included are methods of treating cancer using the bispecific antibodies and oncolytic viruses that encode them.

Description

Oncolytic virus expressing anti-ROR1/anti-CD3 bispecific antibody

The present invention provides bispecific antibodies that simultaneously bind to both ROR1 and CD3. The present invention provides anti-ROR1/anti-CD3 bispecific antibodies, nucleic acids encoding anti-ROR1/anti-CD3 bispecific antibodies, oncolytic viruses including constructs encoding anti-ROR1/anti-CD3 bispecific antibodies, and methods for treating cancer method.

Receptor tyrosine kinase orphan receptors 1 and 2 (ROR1 and ROR2) have been described to be specifically associated with specific cancers (Rebagay et al., 2012, Front Oncol ., 2(34)), while with rare exceptions , which is largely absent from healthy tissue (Balakrishnan et al., 2017, Clin . Cancer Res . , 23(12), 3061-3071). Because of this tumor-selective expression of ROR family members, they represent relevant targets for targeted cancer therapy.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is abnormally expressed in B-cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). ROR1 is almost 100% associated with chronic lymphocytic leukemia (CLL) (Cui et al., 2016, Blood , 128(25), 2931), and has been identified as a key factor in some acute lymphoblastic leukemia (ALL), mantle cell A marker for lymphoma and some other hematological malignancies. ROR1 is also expressed in certain solid tumors, such as those of lung and breast cancer (Balakrishnan et al., 2017, Clin . Cancer Res . , 23(12), 3061-3071). ROR1 has been found to be associated with the progression of many solid tumors, such as neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colorectal cancer, head and neck cancer, and melanoma, and has been shown to inhibit apoptosis and enhance EGFR signaling , Induce epithelial-mesenchymal transition (EMT) and promote cell pit formation.

ROR1 is primarily detectable in embryonic tissues and is generally absent in adult tissues, making the protein an ideal drug target for cancer therapy. Therefore, ROR1 is considered to be a target for the development of ROR1-specific antibodies. However, due to the high homology of ROR1 between different mammalian species, it is 100% conserved in amino acids between humans and cynomolgus monkeys, and 96.7% homologous between humans and mice And there is 96.3% homology between humans and rabbits, it is difficult to produce high-affinity antibodies against this target by standard techniques such as animal immunization.

Oncolytic viruses are viruses that selectively infect and lyse cancer cells. Oncolytic viruses have been the subject of clinical trials for the treatment of various cancers, including melanoma, glioma, head and neck cancer, ovarian cancer, lung cancer, liver cancer, bladder cancer, prostate cancer and pancreatic cancer (Aghi & Martuza (2005) Oncogene 24:7802-7816). Multiple clinical trials have shown that the safety of oncolytic herpes simplex virus (HSV) is compromised by its ability to replicate in normal cells by deleting at least one copy of the gene encoding ICP34.5 (Rampling et al., (2000) Gene Therapy 7: 859-866; Papanastassiou et al., (2002) Gene Therapy 9:398-406; Makie et al., (2001) Lancet 357:525-526; Markert et al., (2000) Gene Therapy 7:867- 874; Markert et al., (2009) Molecular Therapy 17:199-207; Senzer et al., (2009) J Clin Oncol 27:5763-5771).

In addition to directly attacking tumors by lysing cancer cells, oncolytic HSV can induce anti-tumor immune responses in patients (Papanastassiou et al., (2002); Markert et al., (2009); Senzer et al., (2009)) , because viral antigens are expressed on infected cancer cells and tumor antigens are released upon lysis of the cancer cells. Viruses also bind to mediators of the innate immune response as part of host recognition of viral infection that elicits an inflammatory response (Hu et al., (2006) Clin Cancer Res . 12:6737-6747). These immune responses to oncolytic viral therapy can provide systemic benefits to cancer patients, leading to suppression of tumors not yet infected with the virus, including metastatic tumors, and preventing disease recurrence.

The present invention describes a bispecific antibody (αROR1/αCD3 Bsp Ab) that simultaneously binds ROR1 and CD3. The construct encoding the αROR1/αCD3 Bsp Ab as described herein was cloned into an oncolytic HSV-1 virus ("Seprehvec") derived from HSV 17 that did not include a functional RL-1 gene. Virus-infected cells were used to generate virus-free cell culture medium (VFCM), which included bispecific antibodies, which were tested for their ability to enhance T cell cytotoxicity against ROR1-expressing tumor cells. The αROR1/αCD3 BspAb disclosed herein showed potent T cell targeting with specific antitumor activity in preclinical studies. Expression of αROR1/αCD3 BspAb by oncolytic Seprehvec HSV can significantly increase the antitumor activity of viral therapy in an antigen-dependent manner.

In a first aspect, the present invention provides a bispecific antibody comprising a first single-chain variable fragment antibody (ScFv) binding to ROR1 and a second single-chain variable fragment antibody (ScFv) binding to CD3, wherein the anti-ROR1 The scFv and the anti-CD3 scFv are joined by a linker. A linker can be, for example, a GS linker, such as, but not limited to, a (G4S)n linker, where n can be an integer from 1-20, such as 1-8. Anti-ROR1/anti-CD3 bispecific antibodies (αROR1/αCD3 BspAbs) can be isolated proteins, and in some instances partially or substantially purified.

The anti-ROR1 scFv of the bispecific antibody may have a heavy chain variable domain (VH) sequence and a light chain variable domain (VL) sequence connected by a linker, such as a (G4S)n linker, and the VH and VL sequences It can be derived from a monoclonal antibody that binds ROR1, eg, human ROR1 protein. For example, the anti-ROR1 scFv of the bispecific antibody may comprise a VH domain sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 , and to SEQ ID NO:1. ID NO:5 has a VH domain sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In another example, the anti-ROR1 scFv of the bispecific antibody may comprise a VH domain sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, and A VH domain sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In another example, the anti-ROR1 scFv of the bispecific antibody may comprise a VH domain sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, and A VH domain sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23.

In another example, the anti-ROR1 scFv of the bispecific antibody may comprise a VH domain sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:52, and A VH domain sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:56; or an anti-ROR1 scFv of a bispecific antibody may comprise a sequence identical to SEQ ID NO:60 A VH domain sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, and at least 95%, at least 96%, at least 97%, at least 98% to SEQ ID NO:64 Or a VH domain sequence with at least 99% identity.

In various embodiments, the anti-ROR1 scFv of the anti-ROR1/anti-CD3 bispecific antibody (αROR1/αCD3 BspAb) as provided herein may have the same expression as SEQ ID NO:9, SEQ ID NO:18 or SEQ ID NO:27 Amino acid sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

In a non-limiting example, the anti-CD3 scFv of the αROR1/αCD3 BspAb provided herein may comprise an anti-CD3 scFv that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32 An anti-CD3 scFv having a heavy chain variable domain and a light chain variable domain that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:33. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:34.

Another aspect provided by the present invention is a nucleic acid construct encoding any of the αROR1/αCD3 BspAbs disclosed herein. The αROR1/αCD3 BspAb encoded by the nucleic acid construct may include a signal peptide at the N-terminus of the bispecific antibody construct, for example, the signal peptide of SEQ ID NO: 28, or any suitable signal peptide. The nucleic acid construct can be a DNA construct comprising a promoter operably linked to the αROR1/αCD3 BspAb coding sequence. As non-limiting examples, the promoter can be the EF1 alpha promoter, CMV promoter (e.g., SEQ ID NO: 42), JeT promoter, RSV promoter, SV40 promoter, CAG promoter, β-actin promoter , HTLV promoter or EF1α/HTLV hybrid promoter (eg SEQ ID NO: 41). The nucleic acid construct may further include a polyadenylation sequence 3' to the BspAb coding sequence, such as the SV40 3' sequence. The nucleic acid constructs can be provided in vectors and, in some instances, can be propagated into recombinant viral genomes.

Another aspect provided by the present invention is a recombinant oncolytic virus comprising a nucleic acid construct comprising a nucleic acid sequence encoding any of the αROR1/αCD3 BspAbs disclosed herein. In various embodiments, the recombinant oncolytic virus is a recombinant herpes simplex virus (HSV), such as an HSV-1 virus, such as one derived from HSV-1 strain 17, HSV-1 strain F, HSV-1 strain KOS, or HSV -1 virus of strain JS1. In some embodiments, a recombinant oncolytic HSV as provided herein that includes a genetic construct for expressing the αROR1/αCD3 BspAb does not include a functional ICP34.5-encoding gene, and in some instances, all or a portion of the ICP34.5-encoding Genes can be deleted. For example, a recombinant oncolytic HSV can be derived from the HSV 17 strain, and a nucleic acid construct encoding the αROR1/αCD3 BspAb can be inserted into the ICP34.5 encoding locus.

In certain embodiments, the recombinant oncolytic virus comprising a nucleic acid construct comprising a nucleic acid sequence encoding αROR1/αCD3 BspAb may further comprise a nucleic acid sequence encoding a cytokine. For example, an oncolytic virus can include a gene encoding an αROR1/αCD3 BspAb and a gene encoding IL-12. In specific examples disclosed herein, the oncolytic virus includes a gene encoding an αROR1/αCD3 BspAb, such as any such gene disclosed herein, and a gene encoding IL-12, for example, at least 95% identical to SEQ ID NO:46 , human IL-12 that is at least 96%, at least 97%, at least 98%, or at least 99% identical. In some examples, an oncolytic virus can include a gene encoding an αROR1/αCD3 BspAb and a gene encoding a different antibody, for example, a scFv that binds a growth factor or a growth factor receptor such as VEGFR2. In specific examples disclosed herein, the oncolytic virus comprises a gene encoding an αROR1/αCD3 BspAb, such as any such gene disclosed herein, and a gene encoding an anti-VEGFR2 scFv, such as at least 95%, at least A VEGFR2 scFV that is 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, an oncolytic virus as disclosed herein encodes 1) an αROR1/αCD3 BspAb; 2) an IL-12 polypeptide, and 3) an anti-VEGFR2 scFv.

Also included are pharmaceutical compositions comprising a recombinant oncolytic virus, which may be a recombinant oncolytic HSV, comprising a genetic construct for expressing the αROR1/αCD3 BspAb and, optionally, one or more additional transgenes, and Pharmaceutically acceptable excipients. Oncolytic viruses can be provided in saline solution, such as PBS, Ringer's solution (Ringer's), or HBSS, and, as non-limiting examples, the formulation can optionally further include one or more preservatives or cryoprotectants such as DMSO or glycerol ). In some embodiments, the concentration of virus in the pharmaceutical composition is at least 10 ⁶ /ml, at least 10 ⁷ /ml, at least 5×10 ⁷ /ml, or at least 10 ⁸ /ml.

Another aspect is a method of treating cancer by administering an oncolytic virus encoding an αROR1/αCD3 BspAb as provided herein, including a pharmaceutical composition as provided herein. An oncolytic virus may include one or more additional transgenes, such as, but not limited to, a gene encoding an IL-12 polypeptide and/or a gene encoding an antibody that binds VEGFR2. A subject may be one diagnosed with cancer, which may be a hematologic cancer or a solid tumor. As non-limiting examples, an individual can be a canine, equine, or primate, and can be a human individual. The oncolytic virus can be an oncolytic HSV, and the administration can be, for example, intravenous, intraarterial, intracavity, intraperitoneal, intratumoral, or peritumoral delivery. For example, oncolytic viruses can be delivered by injection, infusion, or via a catheter. Such methods may involve multiple administrations, where the administrations may be separated by days, weeks or months.

Also provided are methods of treating an individual using a VFCM produced by culturing cells infected with any of the oncolytic viruses disclosed herein.

The present invention also provides a host cell infected with an oncolytic virus as provided herein, the oncolytic virus comprising a genetic construct for expressing the αROR1/αCD3 BspAb. A host cell can be, for example, a mammalian host cell and can have a cell strain. In some embodiments, the host cells are Vero cells, BHK cells or HEK293 cells. Also provided are methods of treating an individual with cancer using a VFCM produced by culturing cells infected with any of the oncolytic viruses disclosed herein. VFCM can be prepared by, for example, centrifugation and filtration of cell supernatant, wherein VFCM can comprise one or more recombinant polypeptides encoded by oncolytic viruses, such as the αROR1/αCD3 BspAb provided herein, and optionally IL-12 and/or anti-VEGFR2 antibodies. In some embodiments, the individual being treated may be a non-human individual.

In another aspect, there is provided a method for producing bispecific antibodies, including producing any of the αROR1/αCD3 bispecific antibodies disclosed herein, by culturing host cells infected with an oncolytic virus to produce the bispecific antibody comprising the bispecific This is achieved by virus-free conditioned cell culture medium (VFCM) for specific antibodies and isolation of bispecific antibodies from the VFCM, the oncolytic virus comprising a genetic construct for expression of bispecific antibodies. A VFCM can include one or more additional polypeptides or antibodies, such as, but not limited to, IL-12 polypeptides and/or antibodies that bind VEGFR2. Also included are pharmaceutical compositions comprising an αROR1/αCD3 bispecific antibody as disclosed herein, and methods of treating an individual suffering from cancer by administering an αROR1/αCD3 bispecific antibody as disclosed herein to the individual. In some embodiments, the methods include treating a subject, such as but not limited to, a non-human subject, with a VFCM that can be prepared from cell culture using, for example, centrifugation and filtration.

This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 63/173,205, filed April 9, 2021, which is hereby incorporated by reference in its entirety.

Throughout this application, reference is made to various publications, patents and/or patent applications. The disclosures of such publications, patents and/or patent applications in their entireties are incorporated by reference into this application in order to more fully describe the prior art to which this invention pertains. definition:

Unless defined otherwise, technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Generally, terms described herein with respect to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry, and hybridization are those well known in the art And commonly used. The methods and techniques provided herein are generally performed according to conventional procedures well known in the art and as described in various general and more specific references that are cited and discussed herein unless otherwise specified. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). A number of basic literatures describe standard antibody production methods, including Borrebaeck (ed.) Antibody Engineering , 2nd ed. Freeman and Company, NY, 1995; McCafferty et al., Antibody Engineering , A Practical Approach IRL , Oxford Press, Oxford, England, 1996; and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ, 1995; Paul (ed.), Fundamental Immunology , Raven Press, NY, 1993; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989) ) Antibodies : A Laboratory Manual Cold Spring Harbor Press, NY; Stite et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif. and references cited therein; Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY, 1986 and Kohler and Milstein Nature 256: 495-497, 1975. All references cited herein are hereby incorporated by reference in their entirety. Enzyme reactions and enrichment/purification techniques are also well known and performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well known and commonly used in the art. Chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and patient treatment can be performed using standard techniques.

The headings provided herein do not limit the various aspects of the invention, which aspects can be understood by reference to the specification as a whole.

Unless otherwise required by the context herein, singular terms shall include pluralities and plural terms shall include the singular. Use of the singular forms "a" and "the" and any combination of words includes plural referents unless expressly and positively limited to one referent.

It should be understood that the use of alternatives herein (eg, "or") shall mean one or both of the alternatives or any combination thereof.

The term "and/or" as used herein shall mean specifically disclosing each of the specified features or components with or without the presence of other features or components. For example, as used herein in phrases such as "A and/or B", the term "and/or" is intended to include "A and B", "A or B", "A" (alone) and "B "(alone). Likewise, the term "and/or" used in phrases such as "A, B, and/or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms "comprises," "including," "has," and "containing," and their grammatical variations, as used herein, are intended to be non-limiting and, therefore, one or more of the list is not excluded and may be substituted Or add to other items listed. It should be understood that whenever the language "comprising" is used herein to describe an aspect, similar aspects described using the terms "consisting of" and/or "consisting essentially of" are also provided.

As used herein, the term "about" refers to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how measured or determined The value or composition, that is, the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more than one standard deviation, according to the practice in the art. Alternatively, "about" or "approximately" can mean up to 10% (ie, ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg can include any value between 4.5 mg and 5.5 mg. Furthermore, especially in terms of biological systems or processes, these terms can mean up to an order of magnitude, or up to 5 times, a value. When a specific value or composition is provided herein, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that specific value or composition.

As used herein, the terms "peptide," "polypeptide," and "protein" and other related terms are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides can contain natural and unnatural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not been cleaved, eg, by a secretory signal peptide or non-enzymatically cleaved at certain amino acid residues. Polypeptides include mature molecules that have undergone cleavage. These terms encompass natural and artificial proteins, protein fragments of protein sequences, and polypeptide analogs such as muteins, variants, chimeric proteins, and fusion proteins, as well as proteins that are post-translationally or otherwise covalently or non-covalently modified. Two or more polypeptides (eg, 3 polypeptide chains) can be associated with each other by covalent and/or non-covalent association to form a polypeptide complex. Conjugation of polypeptide chains may also involve peptide folding. Thus, depending on the number of polypeptide chains forming the complex, polypeptide complexes may be dimeric, trimeric, tetrameric or higher order complexes.

As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" and other related terms are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant forms as well as chemically synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (such as mRNA), analogs of DNA or RNA produced using nucleotide analogs (such as peptide nucleic acids and non-naturally occurring nucleotide analogs), and its hybrids. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, a nucleic acid molecule of the invention comprises a contiguous open reading frame encoding an antibody or fragment or scFv, derivative, mutein or variant thereof. In one embodiment, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides. Nucleic acids encoding bispecific antibodies are described herein.

The terms "recover/recovery/recovering" and other related terms refer to obtaining a protein (eg, an antibody or antigen-binding portion thereof) from a host cell culture medium or from a host cell lysate or from a host cell membrane. In one embodiment, the protein is expressed by the host cell as a recombinant protein fused to a secretory signal peptide (leader sequence) sequence that mediates the release of the expressed protein from the host cell (e.g., a mammalian host cell). secretion. Secreted proteins can be recovered from the host cell culture medium. In one embodiment, the protein is expressed by the host cell as a recombinant protein without a secretion signal peptide sequence that can be recovered from host cell lysates. In one embodiment, the protein is expressed by the host cell as an membrane-bound protein that can be recovered using detergent to release the expressed protein from the host cell membrane. In one embodiment, regardless of the method used to recover the protein, the protein can be subjected to a procedure to remove cellular debris from the recovered protein. For example, the recovered protein can be subjected to chromatography, gel electrophoresis and/or dialysis. In one embodiment, the chromatography comprises any one procedure or any combination of two or more procedures including affinity chromatography, hydroxyapatite chromatography, ion exchange chromatography, reverse phase chromatography and/or or silica chromatography. In one embodiment, the affinity chromatography comprises protein A or G (from a cell wall component of Staphylococcus aureus ).

The term "isolated" means that the protein (eg, antibody or antigen-binding portion thereof) or polynucleotide is substantially free of other cellular material. A protein can be rendered substantially free of naturally associated components (or components associated with cellular expression systems or chemical synthesis methods used to produce antibodies) by isolation using protein purification techniques well known in the art. The term isolated also means that the protein or polynucleotide is substantially free of other molecules of the same species, eg, other proteins or polynucleotides having different amino acid or nucleotide sequences, respectively. The purity or homogeneity of the desired molecule can be analyzed using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrometry. In various embodiments, bispecific antibodies of the invention are isolated.

The terms "signal peptide", "[peptide] signal sequence", "leader sequence", "leader peptide" or "secretion signal peptide" refer to a peptide sequence located at the N-terminus of a polypeptide. The leader sequence directs the polypeptide chain into the cell's secretory pathway and can direct the integration and anchoring of membrane proteins into the lipid bilayer of the cell membrane. A leader sequence is usually about 10 to about 60 amino acids, more usually about 15 to about 50 amino acids in length. The leader sequence can direct the transport of the precursor polypeptide from the cytosol to the endoplasmic reticulum. In various embodiments, the leader sequence includes a signal sequence comprising a CD8α, CD28 or CD16 leader sequence or a mouse or human Igγ secretion signal peptide. In one embodiment, the leader sequence comprises the murine Igγ leader peptide sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 28).

As used herein, "antigen-binding protein" and related terms refer to a protein comprising a moiety that binds to an antigen and optionally a backbone or framework portion that allows the antigen-binding moiety to employ a protein that facilitates binding of the antigen-binding protein to the antigen. The configuration. Examples of antigen binding proteins include antibodies, antibody fragments (eg, antigen-binding portions of antibodies), antibody derivatives, and antibody analogs. Antigen binding proteins may comprise, for example, alternative protein backbones or artificial backbones with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein; and fully synthetic scaffolds comprising, eg, biocompatible polymers. See eg Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Vol. 53, No. 1: 121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptibody-antibody mimics ("PAMs") and antibody-mimetic-based scaffolds utilizing fibronectin components can be used as scaffolds.

An antigen binding protein may have, for example, the structure of a naturally occurring immunoglobulin. In one embodiment, "immunoglobulin" refers to a tetrameric molecule composed of the same two pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "J" region of about 10 or more amino acids. "D" zone. See generally Fundamental Immunology, Chapter 7 (Paul, W. ed., 2nd ed., Raven Press, N.Y. (1989)) (herein incorporated by reference in its entirety for all purposes). The heavy and/or light chain may or may not include a leader sequence for secretion. The variable regions of each light chain/heavy chain pair form the antibody combining site, such that an intact immunoglobulin has two antigen binding sites. In one embodiment, an antigen binding protein may be a synthetic molecule that has a structure that differs from a tetrameric immunoglobulin molecule but still binds one target antigen or binds two or more target antigens. For example, synthetic antigen binding proteins may comprise antibody fragments, 1-6 or more polypeptide chains, asymmetric polypeptide assemblies, or other synthetic molecules. In various embodiments, bispecific antibodies of the invention exhibit immunoglobulin-like properties and specifically bind to two different target antigens (ROR1 and CD3).

The variable regions of immunoglobulin chains exhibit the same general structure of relatively highly conserved framework regions (FRs) joined by three hypervariable regions (also called complementarity determining regions or CDRs). From N-terminus to C-terminus, both light and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

One or more CDRs can be incorporated into a molecule either covalently or non-covalently to render it an antigen binding protein. Antigen binding proteins may incorporate CDRs as part of a larger polypeptide chain, may have CDRs covalently linked to another polypeptide chain, or may incorporate CDRs non-covalently. CDRs allow the specific binding of an antigen binding protein to a particular antigen of interest.

The amino acid assignment of each structural domain is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991 Definition ("Kabat ID"). Other numbering systems for amino acids in immunoglobulin chains include IMGT.RTM. (international ImMunoGeneTics information system; Lefranc et al., Dev . Comp . Immunol . 29:185-203; 2005) and AHo (Honegger and Pluckthun, J . Mol . Biol . 309(3):657-670; 2001); Chothia (Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948); Contact (Maccallum et al., 1996 Journal of Molecular Biology 262:732-745) and Aho (Honegger and Pluckthun 2001 Journal of Molecular Biology 309:657-670).

As used herein, "antibody/antibodies" and related terms refer to intact immunoglobulins or antigen-binding portions thereof that specifically bind to an antigen. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding moieties include, inter alia, Fab, Fab', F(ab') ₂ , Fv, domain antibody (dAb) and complementarity determining region (CDR) fragments, single chain antibody (scFv), chimeric antibody, diabody, trifunctional Antibodies, tetrafunctional antibodies and polypeptides, these polypeptides contain at least a portion of immunoglobulins sufficient to achieve specific antigen binding to the polypeptides.

Antibodies include recombinantly produced antibodies and antigen binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies. Antibodies include monospecific, multispecific (eg, bispecific, trispecific, and higher specificities). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, and heavy chain dimers. Antibodies include F(ab') ₂ fragments, Fab' fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragments (scFv), camelized antibodies, affibodies, disulfide-linked Fv (sdFv), anti-idiotype antibodies (anti-Id), miniature Antibody. Antibodies include monoclonal and polyclonal populations. In some embodiments described herein, the bispecific antibody comprises two single chain variable fragment antibodies joined by a linker, which may be described as the "scFv portion" or simply "scFv" of the bispecific antibody molecule .

As used herein, "antigen-binding domain", "antigen-binding region" or "antigen-binding site" and other related terms refer to the portion of an antigen-binding protein that contains a protein that interacts with an antigen and contributes to the specificity of the antigen-binding protein for the antigen and Amino acid residue (or other moiety) of affinity. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains. Provided herein are antigen binding domains from monoclonal antibodies and bispecific antibodies.

As used herein, the terms "specific binding", "specifically binds" or "specifically binds" and other related terms in reference to an antibody or antigen binding protein (e.g., a heterodimeric antibody) or antibody fragment The context refers to preferential binding to an antigen in a non-covalent or covalent manner relative to other molecules or moieties (eg, an antibody specifically binds to a particular antigen relative to other available antigens). In one embodiment, if the antibody is 10 ^{- 5} M or less, or 10 ^{- 6} M or less, or 10 ^{- 7} M or less, or 10 ^{- 8} M or less, or 10 ^{- 9} M or If the dissociation constant _KD of 10 ^{- 10} M or less, or 10 ^{- 11} M or less binds to the target antigen, then the antibody specifically binds to the antigen. Described herein are bispecific antibodies that specifically bind ROR1 and CD3.

In one example, binding specificity can be measured by ELISA, radioimmunoassay (RIA), electrochemiluminescence assay (ECL), immunoradiometric assay (IRMA), or enzyme immunoassay (EIA).

In one embodiment, the dissociation constant (K _D ) can be measured using BIACORE surface plasmon resonance (SPR) analysis. Surface plasmon resonance refers to an optical phenomenon that allows the analysis of real-time interactions by detecting changes in protein concentration within a biosensing matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) .

As used herein, "epitope" and related terms refer to the portion of an antigen that is bound by an antigen-binding protein, such as an antibody or antigen-binding portion thereof. An epitope may comprise portions of two or more antigens bound by an antigen binding protein. An epitope may comprise one antigen or non-contiguous portions of two or more antigens (e.g. not contiguous in the primary sequence of the antigen, but sufficiently close together in the case of the tertiary and quaternary structure of the antigen to be bound by the antigen. protein-binding amino acid residues). In general, the variable regions (specifically, the CDRs) of antibodies interact with epitopes. Described herein are bispecific antibodies that bind an epitope of a ROR1 polypeptide and that bind an epitope of a CD3 polypeptide.

As used herein, "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody" or "antigen-binding portion of an antibody" and other related terms refer to molecules that are not intact antibodies, including those that are not related to the intact antibody. The bound antigen-binding portion. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; Fd; and Fv fragments, and dAb; diabodies; linear antibodies; For example, scFv); a polypeptide comprising at least a portion of an antibody sufficient to achieve specific antigen binding to the polypeptide. Antigen-binding portions of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include Fab, Fab', F(ab') ₂ , Fv, domain antibody (dAb) and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies and polypeptides, among others , the polypeptides contain at least a portion of an immunoglobulin sufficient to impart antigen-binding properties to the antibody fragment.

The terms "Fab", "Fab fragment" and other related terms refer to a protein comprising the light chain variable region (V _L ), the light chain constant region ( _CL ), the heavy chain variable region (V _H ) and the first constant region ( A monovalent fragment of _CH1 ). Fab is capable of binding antigen. F(ab') ₂ fragments are bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region. F(ab') ₂ has antigen-binding ability. The Fd fragment includes _VH and _CH1 regions. The Fv fragment comprises _VL and _VH regions. Fv can bind antigen. dAb fragments having _VH domains, _VL domains, or antigen-binding fragments of _VH or _VL domains (US Patents 6,846,634 and 6,696,245; US Published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et al., Nature 341:544-546, 1989).

Single-chain antibodies (scFv) are antibodies in which the VL and _VH regions are linked via a linker (eg, a synthetic sequence of amino acid residues) to form _a continuous protein chain. Preferred linkers are of sufficient length to allow the protein chain to fold upon itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Described herein are single chain antibodies that specifically bind ROR1 and single chain antibodies that specifically bind CD3.

Diabodies are bivalent antibodies comprising two polypeptide chains, where each polypeptide chain comprises a domain that is too short to allow pairing between two domains on the same chain, thus allowing each domain to be complementary to that on the other polypeptide chain. _VH and _VL domains joined by domain-paired linkers (see, eg, Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994 Structure 2:1121-23). If the two polypeptide chains of the diabody are the same, the diabody produced by their pairing will have two identical antigen-combining sites. Polypeptide chains with different sequences can be used to generate diabodies with two different antigen binding sites. Similarly, triabodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and form three and four antigen binding sites, respectively, which may be the same or different.

The term "human antibody" refers to an antibody having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (eg, fully human antibodies). Such antibodies can be prepared in a variety of ways, examples of which are described below, including via recombinant methods or via immunization with an antigen of interest in mice that has been genetically modified to express a protein derived from a human heavy chain and/or Antibodies to light chain encoding genes.

A "humanized" antibody refers to an antibody whose sequence differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions and/or additions such that when the humanized antibody is administered to a human individual , which are less likely to induce an immune response and/or induce a less severe immune response than non-human species antibodies. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of antibodies from non-human species are mutated to generate humanized antibodies. In another embodiment, constant domains from human antibodies are fused to variable domains from a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when the antibody is administered to a human subject. wherein the altered amino acid residues are not critical for the immunospecific binding of the antibody to its antigen, or the amino acid sequence changes are conservative such that the binding of the humanized antibody to the antigen is not significantly inferior to that of a non-human Antibody-antigen binding. Examples of how to make humanized antibodies can be found in US Patent Nos. 6,054,297, 5,886,152 and 5,877,293.

The term "chimeric antibody" and related terms as used herein refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies. In one embodiment, one or more CDRs are derived from a human antibody. In another embodiment, all CDRs are derived from human antibodies. In another embodiment, CDRs from more than one human antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody can comprise CDR1 from the light chain of a first human antibody, CDR2 and CDR3 from the light chain of a second human antibody, and CDRs from the heavy chain of a third antibody. In another example, the CDRs are derived from different species, such as human and mouse, or human and rabbit, or human and goat. Those skilled in the art will appreciate that other combinations are possible.

Furthermore, the framework regions may be derived from one of the same antibodies, from one or more different antibodies (such as human antibodies), or from humanized antibodies. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical, homologous, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to an antibody from another antibody class or subclass. Antibodies of one species or belonging to another antibody class or subclass are identical, homologous or derived therefrom. Fragments of such antibodies that exhibit the desired biological activity (ie, the ability to specifically bind the target antigen) are also included.

As used herein, the terms "variant" polypeptide and polypeptide "variant" refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues differ from a reference polypeptide sequence. Insertions, deletions and/or substitutions. Polypeptide variants include fusion proteins. In the same way, a variant polynucleotide comprises a nucleotide sequence in which one or more nucleotides are inserted, deleted and/or substituted relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

As used herein, the term polypeptide "derivative" is a polypeptide that has been chemically modified, e.g., by conjugation with another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation ( For example, antibodies). Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments and muteins thereof, examples of which are described below.

As used herein, the term "Fc" or "Fc region" refers to the portion of the heavy chain constant region of an antibody that begins or follows the hinge region and terminates at the C-terminus of the heavy chain. The Fc region comprises at least a portion of the CH and CH3 regions, and may or may not include a portion of the hinge region. Two polypeptide chains, each carrying half of an Fc region, can dimerize to form an Fc region. The Fc region can bind to Fc cell surface receptors and some proteins of the immune complement system. The Fc region exhibits effector functions, including any one activity or any combination of two or more activities, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis ( ADP), phagocytosis and/or cell binding. The Fc region can bind Fc receptors, including FcyRI (eg, CD64), FcyRII (eg, CD32), and/or FcyRIII (eg, CD16a).

The term "labeled antibody" or related terms as used herein refers to antibodies and antigen binding portions thereof that are unlabeled or linked to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive , colorimetric, antigenic, enzymes, detectable beads such as magnetic or electron dense (eg gold) beads, biotin, streptavidin or protein A. A variety of labels can be used including, but not limited to, radionuclei species, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (eg, biotin, haptens). Any of the bispecific antibodies described herein can be unlabeled or can be linked to a detectable label or moiety.

As used herein, "percent identity" or "percent homology" and related terms refer to a quantitative measure of the similarity between two polypeptides or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids shared between the two polypeptide sequences at aligned positions, taking into account the possible differences in order to optimize the alignment of the two polypeptide sequences. The number of slots to be introduced and the length of each slot. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides shared between the two polynucleotide sequences at aligned positions, this function taking into account the Optimizing the alignment of polynucleotide sequences may require the number of gaps introduced and the length of each gap. The comparison of sequences and the determination of percent identity between two polypeptide sequences or between two polynucleotide sequences can be accomplished using mathematical algorithms. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences can be determined by using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)), determined by comparing sequences using their preset parameters. Expressions such as "comprising a sequence having at least X% identity to Y" with respect to a test sequence mean that the test sequence comprises a sequence identical to at least X% of the residues in Y when aligned with sequence Y as described above. Residues.

In one embodiment, the amino acid sequence of the test antibody may be similar to but not identical to any amino acid sequence of the polypeptide comprising the bispecific antibody described herein. For any polypeptide comprising a bispecific antibody described herein, the similarity between the test antibody and the polypeptide may be at least 95%, or at least 96% identical, or at least 97% identical, or at least 98% identical, or At least 99% agreement. In one embodiment, similar polypeptides may contain amino acid substitutions within the heavy and/or light chains. In one embodiment, the amino acid substitutions comprise one or more conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity can be adjusted up to correct for the nature of the conservative substitutions. Means for making this adjustment are well known to those skilled in the art. See, eg, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference in its entirety. Examples of groups of amino acids having chemically similar side chains include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxy Side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; ( 5) Basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and formazan Thiamine.

Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins with different antigen specificities. If such antibodies are subjected to affinity purification, they can be enriched for specific antigen specificities. Such enriched antibody preparations are typically made with less than about 10% of the antibodies having specific binding activity for a particular antigen. Subjecting these preparations to several rounds of affinity purification increases the proportion of antibodies with specific binding activity against the antigen. Antibodies produced in this manner are often referred to as "monospecific". Monospecific antibody preparations can be composed of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% % composition of antibodies that have specific binding activity against a particular antigen. Antibodies can be produced using recombinant nucleic acid techniques as described below.

As used herein, "vector" and related terms refer to a nucleic acid molecule (such as DNA or RNA) operably linked to foreign genetic material (such as a nucleic acid transgene). A vector can be used as a vehicle for introducing foreign genetic material into a cell, such as a host cell. The vector may include at least one restriction endonuclease recognition sequence to insert the transgene into the vector. The vector may include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to facilitate selection of host cells bearing the vector-transgene construct. Vectors can be single- or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. The donor nucleic acid used in gene editing methods using zinc finger nucleases, TALENs, or CRISPR/Cas can be a type of vector. One type of vector is a "plastid," which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be ligated to a transgene and is capable of replicating, transcribing and/or translating the transgene in a host cell. Viral vectors typically contain viral RNA or DNA backbone sequences that can be ligated to a transgene. The viral backbone sequence can be modified to disable infection, but maintain the viral backbone and co-linked transgene insertion into the host cell genome. Examples of viral vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, baculovirus, papovavirus, vaccinia virus, herpes simplex virus, and Epstein Barr virus vectors. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell when introduced into the host cell and are thereby replicated together with the host genome.

An "expression vector" is a type of vector that may contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. Expression vectors may include ribosome binding sites and/or polyadenylation sites. An expression vector may include one or more origins of replication sequences. The regulatory sequences direct the transcription, or transcription and translation, of the transgene to which the expression vector is linked for transduction into the host cell. Regulatory sequences can control the amount, timing and/or location of transgene expression. The regulatory sequence can exert its effect on the transgene, for example, directly or through the action of one or more other molecules, such as polypeptides that bind the regulatory sequence and/or nucleic acid. Regulatory sequences can be part of the vector. Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-3606. An expression vector may comprise nucleic acid encoding at least a portion of any of the bispecific antibodies described herein.

A transgene is "operably linked" to a vector when the link between the transgene and the vector permits the function or expression of the transgene sequences contained in the vector. In one embodiment, a transgene is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (eg, amount, timing, or location) of the transgene.

The term "transfection" or "transformation" or "transduction" or other related terms as used herein refers to a method of transferring or introducing exogenous nucleic acid (eg, a transgene) into a host cell. A "transfected" or "transformed" or "transduced" host cell is a host cell that has been transfected, transformed or transduced with an exogenous nucleic acid (transgene). Host cells include primary host cells and their progeny. Exogenous nucleic acid encoding at least a portion of any of the bispecific antibodies described herein can be introduced into a host cell. An expression vector comprising at least a portion of any of the bispecific antibodies described herein can be introduced into a host cell, and the host cell can express a polypeptide comprising at least a portion of the bispecific antibody.

The term "host cell" or "host cell population" or related terms as used herein refers to a cell (or population thereof) into which foreign (exogenous or transgenic) nucleic acid has been introduced. The foreign nucleic acid can include an expression vector operably linked to the transgene, and the host cell can be used to express the nucleic acid and/or the polypeptide encoded by the foreign nucleic acid (transgene). Host cells (or populations thereof) can be cultured cells or can be extracted from an individual. A host cell (or population thereof) includes the primary individual cell and its progeny (regardless of the number of passages). Progeny cells may or may not contain the same genetic material as the parent cells. A host cell encompasses progeny cells. In one embodiment, a host cell describes any cell (including progeny thereof) that has been modified, transfected, transduced, transformed, and/or manipulated in any way to express an antibody, as disclosed herein. In one example, an expression vector described herein operably linked to a nucleic acid encoding a desired antibody, or antigen-binding portion thereof, can be introduced into a host cell (or population thereof). Host cells and populations thereof may contain expression vectors stably integrated into the host genome or may contain extrachromosomal expression vectors. In one embodiment, host cells and populations thereof may contain extrachromosomal vectors that exist after several cell divisions, or extrachromosomal vectors that are transient and disappear after several cell divisions.

Transgenic host cells can be prepared using non-viral methods, including well-known designer nucleases, including zinc finger nucleases, TALENS or CRISPR/Cas. Genome editing techniques such as zinc finger nucleases can be used to introduce transgenes into the genome of host cells. Zinc finger nucleases comprise a pair of chimeric proteins, each containing a non-specific endonuclease of a restriction endonuclease (e.g., FokI) fused to a DNA binding domain from an engineered zinc finger motif enzyme domain. DNA binding domains can be engineered to bind specific sequences in the host genome, and endonuclease domains to form double-stranded cuts. The donor DNA carries a transgene, such as any of the nucleic acids encoding the CAR or DAR constructs described herein, and flanking sequences homologous to regions in the host genome flanking the intended insertion site. The DNA repair mechanism of the host cell can precisely insert the transgene through homologous DNA repair. Zinc finger nucleases have been used to produce transgenic mammalian host cells (US Pat. Nos. 9,597,357, 9,616,090, 9,816,074, and 8,945,868). Transgenic host cells can be prepared using TALENs (transcription activator-like effector nucleases) similar to zinc finger nucleases because they include a non-specific endonuclease domain fused to a DNA binding domain to provide precise Transgene insertion. Like zinc finger nucleases, TALENs also introduce double-stranded cuts into the host's DNA. Transgenic host cells can be prepared using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats; Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR uses a Cas endonuclease coupled to a guide RNA for target-specific donor DNA integration. The guide RNA includes a conserved polynucleotide containing the original spacer-adjacent motif (PAM) sequence upstream of the gRNA-binding region in the target DNA, and a host cell target site that cleaves the double-stranded target DNA with the Cas endonuclease hybrid. Guide RNAs can be designed to hybridize to specific target sites. Similar to zinc finger nucleases and TALENs, the CRISPR/Cas system can be used to introduce site-specific insertions of donor DNA with flanking sequences homologous to the insertion site. Examples of CRISPR/Cas systems for modifying gene bodies are described, eg, in US Patent Nos. 8,697,359, 10,000,772, 9,790,490 and US Patent Application Publication No. US 2018/0346927. In one embodiment, transgenic host cells can be prepared using zinc finger nucleases, TALENs, or CRISPR/Cas systems, and the host target can be the TRAC gene (T cell receptor alpha constant). The donor DNA described herein can include, for example, nucleic acid encoding any of the CAR or DAR constructs. Electroporation, nucleofection, or lipofection can be used to co-deliver donor DNA into host cells with zinc finger nucleases, TALENs, or CRISPR/Cas systems.

The host cell can be a prokaryote, such as E. coli, or it can be a eukaryote, such as a unicellular eukaryote (such as yeast or other fungi), a plant cell (such as a tobacco or tomato plant cell), a mammalian cell (such as a human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells) or fusion tumors. In one embodiment, an expression vector operably linked to a nucleic acid encoding the desired antibody can be introduced into the host cell, thereby producing a transfected/transformed host cell, where a host cell suitable for transfection/transformation The host cells are cultured under conditions to express the antibody and optionally recovered from the transfected/transformed host cells (eg, from host cell lysates) or from the culture medium. In one embodiment, the host cell comprises a non-human cell, including CHO, BHK, NSO, SP2/0, and YB2/0. In one embodiment, the host cells comprise human cells, including HEK293, HT-1080, Huh-7, and PER.C6. Examples of host cells include the COS-7 strain of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary ( CHO) cells or their derivatives, such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), or the DHFR-deficient CHO strain DX-B 11 (see Urlaub et al. Human, 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells (HeLa cells), BHK (ATCC CRL 10) cell line, derived from African green monkey kidney cell line CV1 (ATCC CCL 70 ) CV1/EBNA cell line (see McMahan et al., 1991, EMBO J. 10:2821); human embryonic kidney cells, such as 293, 293 EBNA or MSR 293; human epidermal A431 cells, human Colo 205 cells, transformed other Primate cell lines, normal diploid cells, in vitro cell lines derived from primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, the host cells include lymphocytes, such as YO, NSO or Sp20. In one embodiment, the host cell is a mammalian host cell rather than a human host cell. Typically, host cells are cultured cells that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "transgenic host cell" or "recombinant host cell" may be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid in the desired amount unless regulatory sequences are introduced into the host cell such that they become operably linked to the nucleic acid. It will be understood that the term host cell refers not only to a particular individual cell, but also to the progeny or potential progeny of such cells. Because certain modifications may arise in the progeny due to, for example, mutations or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term as used herein. A host cell or population of host cells as described herein carrying a vector (eg, an expression vector) operably linked to at least one nucleic acid encoding one or more bispecific antibodies.

Polypeptides (eg, antibodies and antigen-binding proteins) of the invention can be produced using any method known in the art. In one example, a polypeptide is produced by a recombinant nucleic acid method by inserting a nucleic acid sequence (eg, DNA) encoding a polypeptide into a recombinant expression vector, introducing the recombinant expression vector into a host cell, and passing the expression vector through the host cell under conditions that promote expression. cell performance.

General recombinant nucleic acid manipulation techniques are described, for example, in Sambrook et al., Molecular Cloning : A Laboratory Manual , Volumes 1-3, Cold Spring Harbor Laboratory Press, 2nd Edition, 1989 or F. Ausubel et al., Current Protocols in Molecular Biology ( Green Publishing and Wiley-Interscience: New York, 1987) and periodically updated, which are incorporated herein by reference in their entirety. A nucleic acid (eg, DNA) encoding a polypeptide is operably linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include transcriptional promoters, optional operator sequences for controlling transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences controlling termination of transcription and translation. An expression vector may include an origin or replication that confers on the host cell the ability to replicate. Expression vectors may include genes that confer selectivity to facilitate recognition of transgenic host cells (eg, transformants).

The recombinant DNA can also encode any type of protein tag sequence that can be used to purify the protein. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Selection and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression vector construct can be introduced into the host cell using methods appropriate to the host cell. Various methods are known in the art for introducing nucleic acid into host cells, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-polydextrose or others; Viral transfection; non-viral transfection; microprojectile bombardment; liposome transfection; and infection (wherein the carrier is an infectious agent). Suitable host cells include prokaryotic cells, yeast, mammalian cells or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms such as Escherichia coli or Bacillus spp . Yeast, preferably from a Saccharomyces species, such as S. cerevisiae , can also be used to produce polypeptides . A variety of mammalian or insect cell culture systems can also be used to express recombinant proteins. A baculovirus system for the production of heterologous proteins in insect cells is reviewed in Luckow and Summers, (Bio/Technology, 6:47, 1988). Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T and BHK cell line. Purified polypeptides are prepared by culturing an appropriate host/vector system to express the recombinant protein. For many applications, the small size of the various polypeptides disclosed herein will make expression in E. coli a preferred method of expression. Subsequently, the protein is purified from the culture medium or cell extract. Any of the bispecific antibodies disclosed herein can be expressed by transgenic host cells.

The antibodies and antigen binding proteins disclosed herein can also be produced using cellular translation systems. For such purposes, the nucleic acid encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA, and to allow cell-free translation of mRNA using systems free of specific cells (eukaryotes, such as those free of mammalian or yeast cells). translation systems, or prokaryotes, such as translation systems without bacterial cells).

Nucleic acids encoding any of the various polypeptides disclosed herein can be chemically synthesized. Codon usage can be selected to improve performance in cells. Such codon usage will depend on the cell type chosen. Specific codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian, plant, yeast and insect cells. See eg: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al., Protein Expr. Purif. 2002(1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al., Microbiol. Rev. 1996 60(3):512-38; and Sharp et al., Yeast. 1991 7(7):657-78 .

Antibodies and antigen-binding proteins described herein can also be produced by chemical synthesis (eg, by the methods described in Solid Phase Peptide Synthesis, 2nd Ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to proteins can also be produced by chemical synthesis.

Antibodies and antigen-binding proteins described herein can be purified by protein isolation/purification methods generally known in the art of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g. with ammonium or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography Chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination of these methods. Following purification, the polypeptides are exchanged in different buffers and/or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

Purified antibodies and antigen binding proteins described herein are preferably at least 65% pure, at least 75% pure, at least 85% pure, more preferably at least 95% pure and most preferably at least 98% pure. Regardless of the exact value of purity, a polypeptide is sufficiently pure to be useful as a pharmaceutical product. Any of the bispecific antibodies described herein can be expressed in transgenic host cells and subsequently purified to a level of about 65-98% purity or higher using any method known in the art.

In certain embodiments, the antibodies and antigen binding proteins herein may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumylation, biotinylation, or addition of polypeptide side chains or hydrophobic groups . Thus, modified polypeptides may contain non-amino acid elements, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which binds one or more sialic acid moieties to the polypeptide. The sialic acid moiety improves solubility and serum half-life, while also reducing the possible immunogenicity of the protein. See Raju et al., Biochemistry. 2001 31;40(30):8868-76.

In one embodiment, the antibodies and antigen binding proteins described herein can be modified to become soluble polypeptides, including linking the antibodies and antigen binding proteins to non-proteinaceous polymers. In one embodiment, the non-proteinaceous polymer is polyethylene glycol ("PEG"), polypropylene glycol, or polyoxyalkylene in the manner described in U.S. Pat. 4,791,192 or 4,179,337.

The present disclosure provides therapeutic compositions comprising any of the bispecific antibodies described herein in admixture with a pharmaceutically acceptable excipient. Excipients encompass carriers, stabilizers and excipients. Examples of pharmaceutically acceptable excipients include, for example, inert diluents or fillers (such as sucrose and sorbitol), lubricants, slip agents, and anti-adherents (such as magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Additional examples include buffers, stabilizers, preservatives, nonionic detergents, antioxidants, and isotonic agents.

Therapeutic compositions and methods for their preparation are well known in the art and are found, for example, in " Remington : The Science and Practice of Pharmacy " (20th Ed., AR Gennaro A R. Ed., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa. ). Therapeutic compositions may be formulated for parenteral administration and may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyethylene oxide-polypropylene oxide copolymers can be used to control the antibody (or its antigen) described herein binding protein) release. Nanoparticle formulations (eg, biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of antibodies (antigen binding proteins thereof). Other potentially suitable parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of antibody (or antigen binding protein thereof) in the formulation will vary depending on a number of factors, including the dose of drug to be administered and the route of administration.

One of the bispecific antibodies (or antigen binding proteins thereof) can optionally be administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid , tartaric acid, methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid or the like; polymeric acids such as tannic acid, carboxymethylcellulose or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid or its analogues. Metal complexes include zinc, iron and similar metal complexes. In one example, the antibody (or antigen binding protein thereof) is formulated in the presence of sodium acetate to enhance thermostability.

Any of the bispecific antibodies (or antigen binding proteins thereof) may be formulated for oral use, including tablets containing the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients. Formulations for oral use may also be presented as chewable tablets, or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or soft gelatin capsules in which the active ingredient is mixed with a water or oil vehicle.

As used herein, the term "individual" refers to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject can be a human, non-human primate, simian, ape, murine (e.g., mouse and rat), bovine, porcine, equine, canine, cat animals, goats, wolves, frogs or fish.

The terms "administering", "administered" and grammatical variants refer to the physical introduction of an agent into an individual using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, eg, by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial , intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal Injection and infusion, and in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route, eg, orally. Other non-parenteral routes include topical, topical or transmucosal administration routes, eg, intranasal, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times and/or over one or more extended cycles. Any of the bispecific antibodies (or antigen binding proteins thereof) described herein can be administered to an individual using methods and delivery routes known in the art.

The terms "effective amount", "therapeutically effective dose" and "effective dose" or related terms are used interchangeably and refer to the amount of an antibody or antigen binding protein (eg bispecific antibody) which, when administered to a subject, is sufficient to Diseases or conditions associated with expression of tumor or cancer antigens result in measurable improvement or prevention. Therapeutically effective amounts of the antibodies provided herein, when used alone or in combination, will vary depending on the relative activities of the antibodies and the combination (e.g., in cytostatic growth inhibition) and will vary depending on the individual and disease condition being treated, the individual The body weight and age and sex of the individual, the severity of the individual's disease condition, the mode of administration and the like, which can be easily determined by one of ordinary skill.

In one embodiment, a therapeutically effective amount will depend on the individual being treated and certain aspects of the condition being treated and can be determined by one skilled in the art using known techniques. Typically, the polypeptide is administered at about 0.01 mg/kg to about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per day, optimally 0.1 mg/kg to about 20 mg/kg per day. Polypeptides can be administered daily (eg, once, twice, three, or four times daily) or more preferably less frequently (eg, weekly, every two weeks, every three weeks, monthly, or quarterly). Additionally, adjustments may be required for age and weight, general health, sex, diet, time of administration, drug interactions and disease severity, as is known in the art.

The present invention provides methods for treating an individual suffering from a disease associated with the expression of one or more tumor-associated antigens, the disease comprising cancer cells or tumor cells expressing tumor-associated antigens such as CD38 and/or CD3 antigens. In one embodiment, the cancer or tumor comprises prostate cancer, breast cancer, ovarian cancer, head and neck cancer, bladder cancer, skin cancer, colorectal cancer, anal cancer, rectal cancer, pancreatic cancer, lung cancer (including non-small cell lung and small cell lung cancer), leiomyoma, brain, glioma, glioblastoma, esophagus, liver, kidney, stomach, colon, cervix, uterus, intrauterine Membrane cancer, vulvar cancer, laryngeal cancer, vaginal cancer, bone cancer, nasal cavity cancer, sinus cancer, nasopharyngeal cancer, oral cavity cancer, oropharyngeal cancer, larynx cancer, lower larynx cancer, salivary gland cancer, urinary tract cancer, urethral cancer, Penile cancer and testicular cancer.

In one embodiment, the cancer comprises hematological cancers, including leukemia, lymphoma, myeloma, and B cell lymphoma. Blood cancers include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitt's lymphoma (BL), B chronic lymphocytic leukemia (B-CLL ), systemic lupus erythematosus (SLE), B and T acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's Macroglobulinemia, mantle cell lymphoma, Hodgkin's Lymphoma (HL) , plasma cell myeloma, precursor B-cell lymphoblastic leukemia/lymphoma, plasma cell tumor, giant cell myeloma, plasma cell myeloma, heavy chain myeloma, light chain or Bence-Jones myeloma myeloma), lymphomatoid granulomatous disease, post-transplantation lymphoproliferative disorder, immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Zeigers disease (Chagas' disease), Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple Sexual sclerosis, antiphospholipid syndrome, ANCA associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, severe chain disease, primary or immune cell-associated amyloidosis, and monoclonal gamma globulinemia of undetermined significance. Recombinant oncolytic virus encoding anti -ROR1 / anti -CD3 bispecific antibody

The present disclosure provides, inter alia, oncolytic viruses expressing bispecific antibodies that bind ROR1 and CD3, cells infected with such viruses, and methods of treating cancer using viruses expressing bispecific antibodies. Also provided are virus-free conditioned medium (VFCM) produced by infected cells and methods of using VFCM to produce pharmaceutical formulations.

Because of their selective replication and lysis in tumor cells, oncolytic viruses provide a targeted approach for cancer therapy. Various types of oncolytic viruses known in the art, including parvovirus, myxoma virus, reovirus, Newcastle disease virus (NDV), Seneca Valley virus (SVV) , poliovirus (PV), measles virus (MV), vaccinia virus (VACV), adenovirus, vesicular stomatitis virus (VSV) and herpes simplex virus (HSV). These viruses replicate in tumor cells and induce cell lysis and/or elicit an immune response to the tumor cells they infect. The present invention provides a recombinant oncolytic virus comprising a heterologous gene construct encoding an anti-ROR1/anti-CD3 bispecific antibody (αROR1/αCD3 BspAb), such as any antibody disclosed herein. The construct may include a promoter active in mammalian cells operably linked to the sequence encoding the αROR1/αCD3 BspAb, and the construct may be inserted into the gene body of an oncolytic virus.

In various embodiments, the oncolytic virus modified to express the αROR1/αCD3 BspAb can be a herpes simplex virus (human alphaherpesvirus; HSV), such as HSV-1, HSV-2 or have HSV-1 or HSV-2 sequences recombinant HSV. For example, laboratory strains or clinical isolates of HSV-1 or HSV-2 strains may be used. A variety of isolated and modified strains of HSV-1 and HSV-2 are known in the art and are contemplated for use in the compositions and methods disclosed herein, including, as non-limiting examples, HSV-1 virus Strain A44, HSV-1 strain Angelotti, HSV-1 strain CL101, HSV-1 strain CVG-2, HSV-1 strain H129, HSV-1 strain HFEM, HSV-1 strain HZT, HSV-1 Virus strain JS1, HSV-1 strain MGH10, HSV-1 strain MP, HSV-1 strain Patton, HSV-1 strain R15, HSV-1 strain R19, HSV-1 strain RH2, HSV-1 virus Strain SC16, HSV-1 strain KOS, HSV-1 strain F and HSV-1 strain 17, HSV-2 strain 186, HSV-2 strain 333, HSV-2 strain B4327UR, HSV-2 strain G, HSV-2 strain G, HSV-2 strain HG52, HSV-2 strain SA8, HSV-2 strain SD90, HSV-2 strain SN03, HSV-2 strain SS01 and HSV-2 strain ST04 . Mutants of these strains, or derivatives of other strains as may be known or isolated in the art, are also contemplated for use in the compositions and methods provided herein.

Derivatives of virus strains include, but are not limited to, viruses that may have: one or more mutated endogenous genes (including partial or complete deletion of one or more endogenous genes), transgenic genes inserted into the viral genome Reproductive genes (heterologous genes) (including, but not limited to, one or more selectable markers, negative selection markers ("suicide genes") and/or detectable markers (such as genes encoding fluorescent proteins or genes encoding The gene for the enzyme that detects the product)), and/or one or more modifications, such as, but not limited to, restriction sites, recombination sites or "landing sites", foreign promoters, and the like. Derivatives may have other modifications, such as, but not limited to, deletions or mutations of non-genic sequences, such as gene regulatory regions such as promoters, or non-coding sequences, such as, but not limited to, direct or inverted repeat sequences. Derivatives of viral strains may be viruses which, alternatively or in addition to other modifications, also include, as non-limiting examples, one or more transgenes that support or regulate viral growth or viability, one or more genes that affect host cell A functional gene, or one or more transgenes encoding a therapeutic protein.

In a non-limiting example, the HSV is HSV-1, such as HSV-1 strain 17, HSV-1 strain KOS or HSV-1 strain F or HSV-1 strain 17, HSV-1 strain KOS Or a derivative of any one of the HSV-1 strains F. For example, the strains used to introduce the ScFv-Fc-TGFβtrap construct can be HSV-1 strain 17 mutant 1716, HSV-1 strain F mutant R3616 (Chou and Roizman (1992) Proc . Natl . Acad 89 : 3266-3270), HSV-1 virus strain F mutant G207 (Toda et al., (1995) Human Gene Therapy 9:2177-2185), HSV-1 virus strain F mutant G47Δ (Todo et al . , (2001) Proc Natl Acad Sci USA 98:6396-6401), HSV-1 mutant NV1020 (Geevarghese et al., (2010) Human Gene Therapy 21:1119-28), RE6 (Thompson et al., (1983) Virology 131:171-179), KeM34.5 (Manservigi et al., (2010) The Open Virology Journal 4:123-156), M032 (Campadelli-Fiume et al., (2011) Rev Med . Virol 21:213-226) , Baco (Fu et al., (2011) Int . J . Cancer 129:1503-10), M032 or C134 (Cassady et al., (2010) The Open Virology Journal 4:103-108) or Thalimogene laherparepvec (“TVec” , formerly OncoVex®; Liu et al., (2003) Gene Therapy 10:292-303) or any other derivative or mutant of these substances.

Mutations in endogenous viral genes may include mutations or deletions in genes that affect viral replication or reproduction in non-cancerous cells, or affect the virus' ability to evade host defenses. For example, an HSV comprising an αROR1/αCD3 BspAb can be deleted in any of the ICP34.5-encoding gene, the ICP6-encoding gene, the ICPO-encoding gene, the vhs-encoding gene, or the ICP27-encoding gene. A mutant that does not produce a functional protein encoded by one or more genes (where the gene is multiplexed) is referred to herein as a gene with a functional deletion. The loss of one or more functions of the ICP34.5 coding gene, ICP6 coding gene, ICPO coding gene and vhs coding gene can lead to impaired replication of HSV in non-cancer cells.

The ICP34.5 encoding gene RL1 is located in the long repeat (RL) of the HSV-1 genome and exists in two copies. In some embodiments one or both copies of the gene encoding ICP34.5 are mutated, or partially or completely deleted, such that no functional protein is produced. In a preferred embodiment, an oncolytic HSV comprising a transgene encoding the ScFv-Fc-TGFβtrap protein and optionally the IL-12 gene functionally deletes the ICP34.5 encoding gene responsible for the neurovirus (Chou et al. , (1990) Science 250:1262-1266), for example, two copies of the ICP34.5 coding gene of the HSV viral genome are inactivated. For example, the oncolytic HSV used to introduce the ScFv-Fc-TGFβtrap construct can be a mutant of HSV-1 strain 17, and can be a mutant of HSV1716 (Brown et al., (1994) Journal of General Virology 75: 2367- 2377; MacLean et al., (1991) Journal of General Virology 72:631-639) or a mutant or a derivative thereof, also Seprehvec™ or a derivative or a mutant thereof. Both HSV1716 and Seprehvec™ have deletions in both copies of the ICP34.5-encoding gene, so they do not produce a functional gene product, but each has a genome substantially similar to HSV strain 17, which has been fully Sequencing (Pfaff et al., (2016) J Gen Virol 97:2732-2741; ncbi.nlm.nih.gov/genome, accession number JN555585).

The recombinant HSV provided herein can have one or more transgenes inserted into the ICP34.5 locus, the ICP6 locus, the ICPO locus, or the vhs locus. In some preferred embodiments, the recombinant oncolytic HSV as provided herein may have an indel of the αROR1/αCD3 BspAb gene in the ICP34.5 coding locus. In some preferred embodiments, the recombinant oncolytic HSV as provided herein has functionally deleted ICP34.5 (i.e. ICP34.5 null), and has two of the αROR1/αCD3 BspAb genes inserted at the ICP34.5 encoding locus copy.

The present invention provides a recombinant oncolytic virus capable of infecting a variety of tumor cell types, including an expression construct encoding a novel bispecific antibody that binds to ROR1 (a protein expressed on many tumor cells) and CD3 (a protein expressed on T cells). ), wherein the bispecific antibody can be expressed and secreted by cells infected with a recombinant virus encoding it. The ROR1 scFv portion of αROR1/αCD3 BspAb specifically binds to immune checkpoint proteins, and the CD3 scFv portion binds to T cells, making T cells adjacent to target tumor cells to promote the killing of tumor cells.

An exemplary construct encoding the αROR1/αCD3 BspAb described herein uses a scFv derived from the ROR1 monoclonal antibody o11, having the heavy chain variable region of SEQ ID NO: 1 or a sequence at least 95% identical thereto, having SEQ ID NO: 1 The heavy chain variable region CDRs of ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and the variable light chain region of SEQ ID NO:5 or a sequence having at least 95% identity thereto, SEQ ID NO :6, the light chain variable region CDRs of SEQ ID NO: 7 and SEQ ID NO: 8.

Another exemplary construct encoding the αROR1/αCD3 BspAb described herein uses a scFv derived from the ROR1 monoclonal antibody s10, having the heavy chain variable region of SEQ ID NO: 10 or a sequence at least 95% identical thereto, Having the heavy chain variable region CDRs of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the variable light chain region of SEQ ID NO: 14 or a sequence at least 95% identical thereto, SEQ ID NO: 14 Light chain variable region CDRs of ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

A further exemplary construct encoding the αROR1/αCD3 BspAb described herein uses a scFv derived from the ROR1 monoclonal antibody jlv1011, having the heavy chain variable region of SEQ ID NO: 19 or a sequence at least 95% identical thereto, having The heavy chain variable region CDRs of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, and the variable light chain region of SEQ ID NO:23 or a sequence having at least 95% identity thereto, EQ ID The light chain variable region CDRs of NO:24, SEQ ID NO:25 and SEQ ID NO:26.

In particular examples, the αROR1/αCD3 BspAb can have the sequence of SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or can have the same sequence as SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO The amino acid sequence of any one of :40 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

The αROR1/αCD3 BspAb can have the structure: heavy chain variable region-linker-light chain variable region or light chain variable region-linker-heavy chain variable region. The anti-ROR1 scFv of the αROR1/αCD3 BspAb can be the N-terminal anti-CD3 scFv portion or vice versa.

Constructs encoding αROR1/αCD3 BspAbs, IL-12 polypeptides, or anti-VEGFR antibodies (eg, anti-VEGFR scFv) are operably linked to promoters for expression in eukaryotic cells. Examples of promoters that can be used to express the αROR1/αCD3 BspAb in recombinant viruses include, but are not limited to, the cytomegalovirus (CMV) promoter (e.g., SEQ ID NO: 33), the hybrid CMV promoter (e.g., U.S. 9,777,290), HTLV Promoter, EF1α promoter, hybrid EF1α/HTLV promoter (eg SEQ ID NO: 32), JeT promoter (US Patent No. 6,555,674), SPARC promoter (eg US 8,436,160), RSV promoter, SV40 promoter Or a retroviral LTR promoter such as the MMLV promoter, or a promoter derived from any of these. Constructs may also include polyadenylation sequences, such as BGH, SV40, HGH or RBG polyadenylation sequences. In some embodiments, the polyadenylation sequence has the sequence of SEQ ID NO:38.

Oncolytic viruses, such as those described herein that include transgenes encoding αROR1/αCD3 BspAb, IL-12, and/or anti-VEGFR antibodies, can be used to infect host cells that can be cultured to produce VFCM, and optionally bispecific Antibodies or other recombinant polypeptides that can be used for therapeutic purposes. VFCMs can be produced using, for example, centrifugation of cell supernatants followed by filtration using, for example, 0.22, 0.2 and/or 0.1 micron filters. Individuals, such as those with cancer, can be treated with a VFCM that includes, for example, an αROR1/αCD3 BspAb. An individual in some embodiments may be a non-human animal, and may be a non-limiting example, a dog, horse, cat, monkey, ape, livestock, or member of an endangered species. The present invention provides methods of treating cancer using recombinant HSV encoding αROR1/αCD3 BspAb. The method can comprise administering to an individual with cancer a recombinant HSV comprising a nucleic acid construct encoding an αROR1/αCD3 BspAb provided herein. In some embodiments, the cancer may be a solid tumor. The recombinant HSV can be any one disclosed herein, such as any one encoding the αROR1/αCD3 BspAb. A subject can be a human or a non-human animal (eg, a dog, cat, cow, bull, or horse). Cancers may be, but are not limited to, bladder, bone, breast, eye, stomach, head and neck, kidney, liver, lung, ovary, pancreas, prostate, skin or uterus, interstitial dermatoma, glioma, neuroblastoma, or chondrosarcoma. Administration can be by any means, and by way of non-limiting examples, can be parenteral, systemic, intracavitary (e.g., intrathoracic, intraperitoneal), peritumoral, or intratumoral, and can be by injection, intravenous or intraarterial infusion, or other delivery methods. Injection can be, for example, parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intratumoral or peritumoral. A treatment regimen may include more than one administration of virus, and may include multiple administrations over days, weeks, or months. In some embodiments, the HSV encoded αROR1/αCD3 BspAb used in the method has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% SEQ ID NO:36, SEQ ID NO:38 or SEQ ID NO: 40 (or a homologous αROR1/αCD3 BspAb with a different or no signal peptide). HSV may further comprise one or more additional transgenes, which may encode, as non-limiting examples, genes having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:47. IL-12 polypeptide or anti-VEGFR scFV having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 49 (or the same with or without a signal peptide) source peptide). Examples Example 1. α - ROR1 / α - CD3 BspAb Herpes Simplex Virus ( HSV ) Construct .

Three constructs were designed for expression of a bispecific antibody (BspAb) comprising from N-terminus to C-terminus: signal peptide (SEQ ID NO:28), scFv antibody specifically binding to ROR1, GS linker (SEQ ID NO:29) and anti-CD3 scFv antibody (hum291, SEQ ID NO:34). Figure 1 provides a general diagram representing a construct encoding an αROR1/αCD3 bispecific antibody (αROR1/αCD3 BspAb). All constructs included the EF1α/HTLV hybrid promoter (SEQ ID NO: 41 ) operably linked to the BspAb coding sequence. Constructs encoding three different αROR1/αCD3 BspAbs differing only in ROR1 scFv were prepared: αCD3/αROR1 BspAb including o11 ROR1 scFv (SEQ ID NO:9), αCD3/αROR1 BspAb including s10 ROR1 scFv (SEQ ID NO:18) αROR1 BspAb and αCD3/αROR1 BspAb including jlv1011 ROR1 scFv (SEQ ID NO:27).

To clone these constructs into the viral genome, BspAb constructs flanked by attL sites were generated by PCR cloning and inserted into the internally deleted RL1 locus of the HSV-1 Seprehvec® genome. Seprehvec® is an HSV-1 vector derived from HSV strain 17 in which both copies of the RL1 gene encoding the γ34.5 kd (ICP34.5) polypeptide responsible for neurotoxicity are deleted by 695 bp (nucleotides within the RL1 sequence 125975 to 125221) to inactivate the RL1 gene. RL1 deletion sites include attR recombination sites for insertion of any gene or construct of interest flanked by attL sequences. Anti-CD3/anti-ROR1 BspAb constructs flanked by attL sequences were inserted into the two RL1 loci at the site of the deletion using in vitro recombination using integrase and LR Clonase™ Plus Enzyme Mix for Integrating Host Factors (ThermoFisher, Carlsbad, CA).

After the recombination reaction, the viral gene body DNA was transfected into BHK (Little Hamster Kidney Fibroblast) cells for the production of recombinant virus. Virus was harvested from transfected BHK cells and subsequently used to infect Vero (African green monkey ( Chlorocebus sp . ) kidney epithelium) cells. Single plaques were harvested from infected Vero cells and passaged into new Vero cells. Repeat this process for a total of four rounds of plaque isolation. Approximately 3.2 x 107 BHK cells were subsequently ^{infected with approximately 3.2 x 105 plaque} forming units (PFU) of virus and cultured for three days to generate virus stocks. After three days, the supernatant was spun twice at 2,100 g to pellet cells from debris. After the cells were pelleted, the supernatant containing the virus was spun at 17,200g to pellet the virus. Virus was resuspended, filtered, and titrated on Vero cells. Virus seed stocks and research stocks were generated from purified αROR1/αCD3 BspAb viruses SepGI-189 (o11 αROR1 scFv), SepGI-201 (s10 αROR1 scFv) and SepGI-203 (jlv1011 αROR1 scFv). Table 1. ROR1 antibody, scFv , bispecific antibody and engineered virus . ROR1 antibody VH Domain SEQ ID NO VL domain SEQ ID NO αROR1 scFv SEQ ID NO αROR1/αCD3 BspAbBspAb (precursor) SEQ ID NO Oncolytic HSV expressing αROR1/αCD3 BspAb o11 1 5 9 36 SepGI-189 s10 10 14 18 38 SepGI-201 jlv1011 19 twenty three 27 40 SepGI-203 Example 2. Generation of virus-free conditioned medium ( VFCM ) from HSV expressing the αROR1 / αCD3 BspAb construct .

To generate virus-free conditioned medium (VFCM) from the αROR1/α-CD3BspAb viruses SepGI-189, SepGI-201 and SepGI-203, 3 _cells were inoculated in 1 mL medium in 12-well plates at 37°C, 5% CO. × ¹⁰⁵ A431 cells or HepG2 cells in a separate dish. The next day, A431 cells and HepG2 cells were infected with recombinant HSV at an MOI (magnification of infection) of 0.5, and cultured in 1.25 mL medium for 3 days. After 3 days, the cell supernatant was removed and filtered through a 0.1 µm membrane (Pall Acrodisc Syringe Filter Part #4611) to remove virus. The VFCM was then aliquoted and stored at -80°C. SepGI-Null, that is, the VFCM of the Seprehvec® HSV vector that does not include the foreign transgene was also prepared as a control. Example 3. Detection of αROR1 / αCD3 bispecific antibody in VFCM .

50 μl/well of recombinant human ROR1 Fc fusion protein was coated on a 96-well plate at a concentration of 2 μg/mL (R&D Systems, catalog number 9490-RO-050). The plates were then sealed and incubated overnight at 4°C. The next day, the plates were washed with 150 μl/well of wash buffer (Dulbecco's phosphate-buffered saline 1X with 0.05% V/V Tween 20). Non-specific binding was blocked using 80 μl/well of blocking buffer (Dulbecco's phosphate buffered saline with 2% BSA + 0.05% Tween20) and the plates were incubated at 37°C for 1 hour. After washing three times, the virus-free medium (VFCM) containing αROR1/αCD3 BspAb of SepGI-189, SepGI-201 or SepGI-203 (VFCM) and the control VFCM (SepGI-Null) were serially diluted in blocking buffer and incubated at room temperature. Incubate at 50 μl/well for 2 hours at (RT) with gentle shaking. Plates were washed three times with wash buffer and 50 μl/well of anti-CD3 Hum291 anti-idiotype clone 5A2 diluted in blocking buffer (1 :80 dilution) was added. Plates were incubated at 37°C for 1 hour. After three washes, 50 microliters/well of goat anti-rabbit IgG HRP antibody (Abcam; cat. no. ab6721 ) diluted 1:120,000 in blocking buffer was added. Plates were incubated for 1 hour at 37°C in the dark. Plates were washed three times with wash buffer, and SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution (SeraCare, cat. no. 5120-0082) was added to the wells (80 microliters/well). Plates were incubated for 10-15 minutes at room temperature in the dark. Signal generation was stopped by adding 50 microliters/well of TMB Blue STOP solution (SeraCare, cat. no. 5150-0022), followed by reading the signal at 450 nm (for SeraCare TMB BlueSTOP solution) using a TecanSpark or other device. Figure 2A provides a schematic of the assay. Figure 2B shows that all three bispecific constructs are expressed by the engineered SepGI oncolytic viruses SepGI-189, SepGI-201 and SepGI-203 and are able to bind ROR1. Example 4. Binding of αROR1 / αCD3 BspAb from VFCM of virus infection medium to ROR1 positive tumor cells .

Knockdown of ROR1 in A549 human alveolar adenocarcinoma (non-small cell lung cancer) cells using a CRISPR/Cas-9 approach. A549/ROR1 knockout (A549/ROR1-KO) cells or A549 wild-type (WT) cells were transferred to V-bottom 96-well dishes (80,000 cells per well). Serial dilutions (1:5 to 1:3, 125-fold dilution) in FACS buffer (PBS 1X + 2% FCS/FBS) of virus-free medium (VFCM) preparations produced as described in Example 2 were added to the wells (100 μl/well) and incubated with the cells for 1 hour at room temperature. After three washes, cells were resuspended in 100 μl/well of monoclonal rabbit anti-CD3 Hum291 anti-idiotype antibody (clone 5A2), diluted at 10 μg/mL in FACS buffer. Cover the dish with a dish seal and incubate at 37°C for 1 hour.

Cells were then resuspended in 100 microliters/well of FACS buffer containing donkey anti-rabbit APC (Southern Biotech; Cat. No. 6441-31-31, Lot No. K2916-Z779B) diluted 1:1000, and plated on Incubate at 37°C for 1 hour in the dark. Finally the cells were resuspended in 120 μl/well of FACS buffer and the signal was analyzed on an AttuneNxt flow cytometer. Figure 3A provides an assay format in which ROR1 expressing A549 WT cells bind to BspAb present in VFCM, which is also recognized by anti-idiotype CD3 antibody. Complexes were visualized with an allophycocyanin (APC)-labeled donkey anti-rabbit antibody. Binding of BspAbs present in VFCM to A549/ROR1-KO cells is not expected to occur. Figure 3B provides flow cytometry results showing that the VFCM of all viruses including the bispecific constructs contained the αROR1/αCD3 bispecific antibody, which bound to A549 tumor cells expressing ROR1, but failed to bind to A549/ROR1-KO cell binding. VFCM prepared from cell cultures infected with a control virus not including the bispecific construct (SepGI-Null) did not contain antibodies capable of binding to cells and anti-idiotypic CD3 antibodies. Example 5. Binding of αROR1 / αCD3 bispecific antibody from VFCM from virus-infected cultures to ROR1- positive tumor cells .

A549-WT and A549-ROR1 KO cells were stained with eFluor450 dye (Thermo Fisher Scientific; Cat# 50-246-096) according to the manufacturer's recommendations. Purified human T cells were freshly isolated from healthy blood donors using the Human PAN T Cell Isolation Kit (Miltenyi Biotec; Cat. No. 130-096-535) and stained with eFluor670 dye (Thermo Fisher Scientific; Cat. No. 65-0840-85) for staining. Cells were resuspended in Dulbecco's 1X phosphate-buffered saline (DPBS) at 37°C at 1.0E+07 cells/ml. Mix eFluor450-labeled A549-WT or A549-ROR1 KO tumor cells with purified eFluor670-labeled T cells at a ratio of 1:1 (30,000 tumor cells and 30,000 T cells/well) in U-shaped bottom low-adhesion 96 wells Plates (in 100 μl/well complete RPMI 1640 medium containing 10% FBS). Cells were centrifuged at 1,500 rpm for 3 minutes, and the supernatant was removed by quickly tapping the disc. Cell pellets were resuspended in 50 μL undiluted virus-free medium (VFCM) containing αCD3/αROR1 bispecific constructs (SepGI-189, SepGI-201 or SepGI-203). Cells were incubated at 37°C for 1 hour. Cells were then fixed with 100 μL of Fixation Buffer (Biolegend; Cat# 420801, Lot# B295965) added directly to the wells and incubated with antibodies in the dark at room temperature without disrupting cells or pipetting. Incubate for 20 minutes. Samples were then analyzed on the Attune NxT Cytometer without washing or pipetting. Figure 4A shows an assay design in which BspAbs that bind both ROR1 (expressed on WT A549 cells) and CD3 (expressed on T cells) are able to bind both eFluor 450-labeled WT A549 cells and eFluor 670-labeled T cells ( but not to A549/ROR1-KO cells).

Figure 4B shows the fluorescent quadrants of labeled T cells (alone) and labeled WT A549 cells (alone) after flow cytometry. The rightmost curve shows that when cells are mixed in the presence of VFCM, cells appear in new regions of the curve, indicating that the two fluorophores are in close spatial contact. The graph shows the percentage of interaction of T cells with WT A549 cells and A549/ROR1-KO cells in VFCMs each including αROR1/αCD3 BspAb constructs, which were induced by SepGI-189 virus, SepGI-201 virus and SepGI-203 Culture medium for virus infection. In each case, the interaction of WT A549 cells with CD3 cells was significantly higher than that of A549/ROR1-KO cells with CD3 cells, indicating that all three bispecific antibodies can interact with tumor and T cells in an antigen-specific manner combined. Example 6. Functionally active αROR1 / αCD3 bispecific antibody of VFCM .

To test the ability of the bispecific αROR1/αCD3 antibody to induce signaling in T cells, an assay was performed using Jurkat cells harboring a luciferase gene under the control of an NFAT response element (Jurkat-NFAT-Luc). Figure 5A depicts an assay setup in which a BspAb bound to ROR1 expressing A549 cells also binds to CD3 on Jurkat cells, resulting in signaling that produces luciferase expression and a luminescence signal. A549/ROR1-KO cells that do not bind the αROR1/αCD3 BspAb do not stimulate Jurkat cell signaling.

For the assay, 20,000 A549-WT or A549-ROR1 KO cells were plated in 100 µL of complete RPMI-1640 (RPMI-1640 with 10% FCS) in white opaque flat-bottomed 96-well assay plates (Corning Cat. No. 3917). The plates were spun at 1,500 rpm for 1 minute and incubated overnight at 37°C for cell attachment. The disc was then spun at 1,500 rpm for 3 minutes, and the supernatant was removed by quickly tapping the disc. The wells were then plated with Jurkat cells expressing luciferase under the control of the NFAT response element (Jurkat-NFAT-Luc) (30,000 cells in 50 µL complete RPMI-1640 medium/well). Cell activation was induced by adding 50 μl/well of αROR1/αCD3 (SepGI-189, SepGI-201 or SepGI-203) or negative control VFCM (SepGI-Null) diluted 1:1,000. Purified anti-CD3 clone Hum291 antibody was added to a separate well at 2 µg/mL as a positive control for T cell activation. Plates were incubated for 5 hours at 37°C in a humidified cell incubator. By adding 100 μl/well of Bio- ^Glo® Luciferase Assay Substrate (Promega; Cat# G7940; Lot# 0000422404) and incubating the plate in the dark for 5 minutes at room temperature with gentle shaking according to the manufacturer's recommendations. Displays a glowing signal. The luminescence signal was read with a TECAN device (integration time: 500 ms). Figure 5B shows that all three VFCMs induce T cell activation, with SepGI-201 and SepGI-203 VFCMs inducing potent T cell activation in a ROR1-dependent manner. Example 7. Cytotoxicity assay using VFCM containing αROR1 / αCD3 bispecific antibody .

To test the killing of ROR1-expressing tumor cells by T cells in the presence of the αROR1/αCD3 bispecific antibody, the following cytotoxicity assay was performed. On day 0, 10,000 A549-FLuc WT and A549-FLuc ROR1 KO cells (target cells) were plated in 100 µL of complete RPMI1640 (supplemented with 10 % FCS of RPMI1640). The plates were spun at 1,500 rpm for 1 minute and incubated overnight at 37°C to allow cells to adhere.

On day 1, human peripheral blood mononuclear cells (hPBMCs) were isolated from human healthy whole blood using a Pan T cell isolation kit (Miltenyi Biotec; Cat. hPBMC to isolate human T cells.

The supernatant was removed from the 96-well plate containing the target cells by quickly tapping the plate, and the VFCM containing the α-ROR1/α-CD3 bispecific antibody was diluted in complete RPMI1640 at 100 μl/well added to target cells. Subsequently, 100 μl/well of purified human T cells (effector cells) (5,000 cells/well) were added to target cells to achieve an E:T ratio of 0.5:1. As a control, some wells received no effector cells. Cells were mixed gently, spun at 1,500 rpm for 1 minute and incubated at 37°C for 3 days. On day 4, the supernatant (100 μl/well) was collected, and Meso Scale Discovery's Pro-inflammatory Panel 1 (Human) Set (MSD; Cat. No. K15049D) was used to measure the inflammatory response under each condition according to the manufacturer's recommendations. IFNγ performance level. Killing activity was assessed by measuring the luminescence signal by adding 100 μl/well of Bio- ^Glo® Luciferase Assay substrate (Promega; Cat. No. G7940; Lot No. 0000422404) according to the manufacturer's recommendations and in Incubate in the dark for 8 minutes at room temperature with gentle shaking to reveal. The luminescence signal was read with a TECAN device (integration time: 500 ms). The percent kill of a sample was calculated as follows: 100-([luminescent _sample /baseline luminescent _{no VFCM control} ])×100.

The results provided in Figure 6 show that VFCM infected with SepGI-189 and SepGI-201 cells can stimulate T cells to kill ROR1-expressing tumor cells, and this effective killing is specific to ROR1-expressing tumor cells. In the presence of αROR1/αCD3 bispecific antibody, T cells co-cultured with target cells also secrete a large amount of interferon γ. Example 8. Cytotoxicity assay involving VFCM containing αROR1 / αCD3 bispecific antibody using tumor lines with different levels of ROR1 expression .

The expression of ROR1 on tumor cell lines A549-Fluc WT, A459-Fluc/ROR1 KO, MCF-7-Fluc and HepG2-Fluc expressing firefly luciferase (Fluc) was evaluated by flow cytometry. Briefly, cells were plated at 80,000 cells/well in a V-bottom 96-well plate and treated with 170 μl/well of FACS buffer (PBS 1X + 2% FCS/FBS + 0.1% sodium azide ) washed twice. Dilute purified human anti-human ROR1 antibody at different concentrations (ranging from 10 to 0.00061 µg/mL; dilution ratio 1:4) in FACS buffer, then resuspend cells in 100 µl/well of diluted antibody , and incubate at 4°C for 30 minutes. After washing twice in 170 μl/well of FACS buffer, the cells were treated with AF647-conjugated goat anti-human IgG secondary antibody (Southern Biotech; Cat. No. 2040-31, Lot No. K471X873C; in FACS buffer at 1: 2,000 dilution) were incubated together at 80 μl/well for 20 minutes at 4°C. Cell pellets were washed twice, then resuspended with 120 µL of fixation buffer (Biolegend; Cat. No. 420801, Lot. No. B306498) and incubated for 15 min at room temperature in the dark. Cells were then centrifuged at 1,500 rpm for 2 minutes, and the supernatant was removed by quickly tapping the disc. Cells were washed twice, resuspended in 150 μl/well of FACS buffer, and collected by flow cytometry on an Attune NxT. Data were analyzed using FlowJo v10. Figure 7A shows that among human tumor cell lines, A549 (alveolar adenocarcinoma) expresses the highest level of ROR1, while HepG2 (liver carcinoma) expresses very little ROR1, and the marker detected by ROR1 antibody is similar to that of A549-ROR1 knockout cells. MCF-7 (breast cancer) cells express intermediate levels of ROR1.

For killing assays, human peripheral blood mononuclear cells (hPBMCs) were isolated from healthy human whole blood and subsequently autosynthesized using the EasySep Human T Cell Isolation Kit (StemCell Technology; Cat# 17951, Lot# 1000024139) according to the manufacturer's recommendations. Human T cells were isolated from hPBMC. A549-Fluc WT, A459-Fluc/ROR1 KO, MCF-7-FLuc, and HepG2-Fluc target cells were plated at 10,000 cells/well in 100 wells of a white opaque flat-bottomed 96-well assay plate (Corning Cat. No. 3917). µL of complete medium. The plates were spun at 1,500 rpm for 1 minute and incubated overnight at 37°C to allow cells to adhere. The supernatant was removed from the 96-well plate containing the target cells by quickly tapping the plate. Purified effector T cells (100 μl/well) were plated on target cells at an E:T ratio of 2:1. As a control, some wells received no effector cells. VFCM from SepGI-201-infected cultures or SepGI-Null-infected cultures was diluted in complete RPMI1640 and added to cells at 100 microliters/well. SepGI-201 virus included the αROR1/αCD3 BspAb construct, and SepGI-Null virus did not include the BspAb construct. Cells were mixed gently, spun at 1,500 rpm for 1 minute and incubated at 37°C for 3 days before collecting the supernatant (100 microliters/well). The level of IFNγ present in the supernatant was measured using the Proinflammatory Panel 1 (Human) Kit (MSD; Lot K15049D) from Meso Scale Discovery according to the manufacturer's recommendations. Killing activity was assessed by measuring the luminescence signal by adding 100 μl/well of Bio- ^Glo® Luciferase Assay substrate (Promega; Cat. No. G7940; Lot No. 0000422404) according to the manufacturer's recommendations and incubating in the laboratory. Incubate in the dark for 5 minutes at room temperature with gentle shaking. The luminescence signal was read with a TECAN device (integration time: 500 ms). The percent kill of a sample was calculated as follows: 100-([luminescent _sample /baseline luminescent _{no VFCM control} ])×100.

Figure 7B shows that although T cells killed A549 and MCF-7 cells in the presence of SepGI-201 VFCM, HepG2 cells were retained even though T cells were activated at high VFCM concentrations (due to low ROR1 expression), as shown by Indicated by increased expression of IFNγ. Example 9. The killing activity of oncolytic viruses SepGI - 189 , SepGI - 201 and SepGI - 203 expressing bispecific antibodies .

Figure 8A provides evaluation of the killing of A549 tumor cells by oncolytic viruses SepGI-189, SepGI-201 and SepGI-203 expressing "o11" αROR1/αCD3, "s10" αROR1/αCD3 and "jlv1011" αROR1/αCD3 BspAb constructs, respectively The experimental plan ( Table 1 ). On day 0, A549-Fluc WT and A549-Fluc ROR1 KO target cells were plated at 10,000 cells/well in 100 µL complete RPMI-1640+10% FCS onto white opaque flat bottom 96-well assay plates (Corning catalog No. 3917). The plates were spun at 1,500 rpm for 1 minute and incubated overnight at 37°C for cell attachment. On day 1, target cells were infected with αROR1/αCD3 virus (SepGI-189, SepGI-201 or SepGI-203, see Table 1 ) or negative control SepGI-Null virus at a multiplicity of infection (MOI) of 1, 0.33, 0.11, 0.04 and 0.01. On day 2, T cells were purified from freshly isolated PBMCs using a Pan T Cell Isolation Kit (Miltenyi Biotec; Cat# 130-096-535, Lot# 519115439) according to the manufacturer's recommendations. The supernatant was removed from the target cells by quickly tapping the 96-well plate, and 20,000 effector T cells were plated at 100 μl/well to achieve a 2:1 E:T ratio. Mix cells gently, spin at 1,500 rpm for 1 min, and incubate at 37°C for 4 days. On day 6, use 100 μl/well of Bio- ^Glo® Luciferase Assay substrate (Promega; Cat# G7940; Lot# 0000422404) according to the manufacturer's recommendations and incubate in the dark at room temperature with gentle shaking for 5 days. Minutes, the killing activity was assessed by measuring the luminescent signal. The luminescence signal was read with a TECAN device (integration time: 500 ms). The percent kill of a sample was calculated as follows: 100-([luminescent _sample /baseline luminous _{virus -free} ])×100.

Figure 8B shows that at an MOI as low as 0.11, the proportion of tumor cells infected with each BspAb-expressing virus was significantly higher than that of tumor cells infected with SepGI-null virus. When the tumor cells were infected with SepGI-201, when the MOI was 0.04, the killing of tumor cells was significantly higher, while when the tumor cells were infected with SepGI-203, when the MOI was 0.01, the killing of tumor cells was significantly higher. The same effect was not seen when viral infection of ROR1 knocked out tumor cells and was used as a target. In this case, only infection with SepGI-203 virus caused significantly higher killing, indicating that SepGI-203 exhibited some degree of non-specific killing at MOI above 0.11. All these data collectively indicate that the combination of oncolytic activity and the αROR1/αCD3 BspAb significantly enhanced antitumor activity in an antigen-specific dependent manner. Example 10. Mouse cross-reactivity between S10 and jlv1011 monoclonal antibody .

To test whether the s10 (RO6D8-s10) and jlv1011 (RO6D8-jlv1011 ) monoclonal antibodies used to engineer the αROR1/αCD3 bispecific antibody also recognized mouse ROR1 in addition to human ROR1, the assay described in Figure 9A was used. Coat 96-well plates at 2 µg/mL with 50 µl/well of recombinant mouse ROR1 IgG ₂ -Fc fusion protein (R&D Systems, Cat. No. 9910-RO-050, Lot No. DIWM0120121), then seal the dish and store at 4°C. Incubate overnight. The next day, the plates were washed with 150 μl/well of wash buffer (Dulbecco's phosphate buffered saline 1X with 0.05% V/V Tween 20). Non-specific binding was blocked using 80 μl/well of blocking buffer (Dulbecco's phosphate buffered saline with 2% BSA + 0.05% Tween20) and the plates were incubated at 37°C for 1 hour. After three washes, two anti-human ROR1 antibodies (s10 and jlv1011 ) were serially diluted (80 μl/well) in blocking buffer and incubated for 2 hours at room temperature (RT) with gentle shaking. The plate was washed three times with wash buffer, and then 80 microliters/well of secondary HRP-labeled goat anti-human IgG Fc (SouthernBiotech; cat. no. 2081-05) diluted in blocking buffer (1:2,000 dilution) was added; Lot No. L5311-TE40). Plates were incubated for 1 hour at 37°C in the dark. Plates were washed three times with wash buffer, and SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution (SeraCare, cat. no. 5120-0082) was added to the wells (80 microliters/well). Plates were incubated for 10-15 minutes at room temperature in the dark. Signal generation was stopped by adding 50 microliters/well of TMB Blue STOP solution (SeraCare, cat. no. 5150-0022), and the signal was then read at 450 nm (for SeraCare TMB BlueSTOP solution) using a TecanSpark or other device. Figure 9B shows that the two anti-ROR1 antibodies used to generate the BspAb constructs of SepGI-201 and SepGI-203 exhibit mouse cross-reactivity. Example 11. In vivo studies of tumor therapy with viruses expressing BspAb .

Figure 10A provides a graph of the vaccination and treatment time course of mice used to test the effectiveness of oncolytic viruses expressing αROR1/αCD3 bispecific antibodies. On day -6 (D-6), female NSG-Tg (Hu-IL-15) mice (6 weeks old) were injected intraperitoneally (IP) with 1.0E+07 freshly purified human peripheral blood mononuclear Dulbecco's Phosphate Buffered Saline (DPBS) 1X for cells (PBMC). On day 0 (D0), mice were injected subcutaneously (SC) in the right flank with 5.0E+06 A549-WT tumor cells diluted in 100 µL DPBS 1X. On day 6 (D6), the mice were randomly divided into four groups (three 'viral-treated' groups and one 'no-viral-treated' control group) and viral treatment was initiated: on days 6, 10, and On day 12, SepG1-189, SepG1-201, SepGI-Null or no virus was delivered peritumorally (PT) at 50 μl/mouse/injection. Tumor growth and body weight were monitored twice weekly. Tumor volume was measured using calipers and calculated using the formula V=(length× ^width2 )/2. The study was terminated on day 31, and the percentage of tumor growth inhibition (TGI) was calculated as follows: [1-(relative tumor volume of the treatment group)/(relative tumor volume of the control group)]×100.

Figures 10B and 10C provide the tumor volume and calculated tumor growth inhibition (TGI) for the treated and non-treated groups, and Figure 10D provides the body weight of the mice during the experiment. Treatment with viruses expressing αROR1/αCD3 bispecific antibodies (SepGI-189 and SepGI-201) elicited greater tumor growth inhibition. Example 12. Constructs for expression of ROR1 / CD3 bispecific antibody and additional gene encoding IL - 12 or IL - 12 plus anti - VEGFR antibody .

Additional constructs were prepared for the synthesis of HSV encoding ROR1-CD3 bispecific antibody and interleukin IL-12. The SepGI-216 construct (dual gene construct, FIG. 11A ) includes the αROR1(s10)-αCD3 bispecific antibody (SEQ ID NO:18) under the control of the EF1α/HTLV promoter (SEQ ID NO:41) and the Gene encoding human IL-12 (SEQ ID NO:46) under the control of the CMV promoter (SEQ ID NO:42). The human IL-12 gene encodes a single polypeptide (SEQ ID NO:47) comprising the p40 and p35 subunits of IL-12 linked by a 2x elastin linker (SEQ ID NO:66). The SepGI-212 construct (three-gene construct, FIG . 11B ) includes the gene encoding the αROR1(s10) -αCD3 bispecific antibody (SEQ ID NO: 37) and the gene (SEQ ID NO: 50) and human IL-12 gene (SEQ ID NO: 46) encoding the anti-VEGFR2 scFv linked to the Fc1 region, wherein the sequence encoding αVEGFR2 scFv-Fc1 (SEQ ID NO: 48) Separated from the sequence encoding human IL-12 (SEQ ID NO:46) by the sequence encoding the T2A self-cleaving peptide (SEQ ID NO:51), encompassing the sequences encoding the human IL-12 polypeptide and the VEGFR2 scFv-Fc1 polypeptide The continuous open reading frame is under the control of the CMV promoter (SEQ ID NO: 42) (see Figure 11B ).

In addition, as a control, a similar construct was designed in which the gene encoding the αROR1(s10)-αCD3 bispecific antibody was encoded to include the scFv binding to the F protein (SEQ ID NO:43) and CD3 of respiratory syncytial virus. Gene replacement of the bispecific antibody (herein referred to as "αRSV-αCD3 bispecific antibody"). The cloning of these constructs and the production and isolation of recombinant viruses were performed substantially as described in Example 1. Table 2. SepGI HSV constructs . _ Virus transgene SepGI-Null none SepGI-201 αROR1(s10)-αCD3 SepGI-207 αRSV-αCD3 SepGI-212 αROR1(s10)-αCD3; αVEGFR2-Fc; huIL-12 SepGI-214 αRSV(s10)-αCD3; αVEGFR2-Fc; huIL-12 SepGI-216 αROR1(s10)-αCD3; huIL-12 SepGI-218 αRSV(s10)-αCD3; huIL-12

Virus-produced VFCMs (see Example 2) were tested to assess transgene expression by ELISA substantially as described in Example 3, with wells of plates coated with ROR1, RSV protein, or human VEGFR.

The results of the ELISA are provided in Figure 12A , Figure 12B and Figure 12C . The first graph ( FIG . 12A ) shows that all three viruses (SepGI-207, SepGI-214, and SepGI-218) that included genes encoding antibodies to the RSV protein produced antibodies to RSV, while the other viruses (without antibodies to the RSV protein) did not produce antibodies . Figure 12B shows that, as expected, viruses SepGI-201, SepGI-216 and SepGI-212 all express ROR1 antibodies, while a control virus without the gene encoding the αROR1(s10)-αCD3 bispecific antibody does not express ROR1 antibodies. Figure 12C shows the results of the ELISA used to detect IL-12. In this case, VFCM from cells infected with SepGI-212, SepGI-216 and SepGI-218 were found to contain IL-12 protein, whereas two isolates of cells infected with SepGI-214 did not contain IL-12 protein. (Isolates of SepGI-214 were subsequently found to produce IL-12.) As expected, IL-12 was not detected in VFCM from uninfected cultures or cultures infected with SepGI Null virus or SepGI-207 virus.

The results of the ELISA detecting the VEGFR2 scFv antibody are shown in Figure 13 , in which the wells of the 96-well ELISA plate are coated with recombinant human VEGFR2 (VEGFR2/ KDR protein (ECD, His tag) (Sino Biological). After antigen wells, VFCM was serially diluted 8-fold in blocking buffer, added at 50 μl/well, and the plate was incubated on a shaker at room temperature for 2 hours. Wash the plate 3 times with washing buffer, and add 50 µl/well of goat anti-human IgG (H+L) secondary antibody, HRP (diluted 1:5,000 in blocking buffer) (Invitrogen), and plates were incubated at 37˚C for 1 hour. After washing the plate with liquid for 3 times, detect the signal with 50 μl/well of SureBlue Reserve TMB 1-component microwell peroxidase substrate solution (catalogue number 5120-0082, SeraCare). The plate was incubated at room temperature in the dark 10-12 minutes. Then add the TMB BlueSTOP solution (catalogue number 5150-0022, SeraCare) of 50 microliters/well, read the absorbance at 450 nm (for the SeraCare TMB BlueSTOP solution) with TecanSpark. The graph in Fig. 13 shows, three The VFCM of an isolate of the gene virus SepGI-212 showed the expression of αVEGFR2-Fc antibody, and the viruses that did not include the αVEGFR2-Fc antibody gene (SepGI-Null, SepGI-201, SepGI-207, SepGI-216 and SepGI-218) Binding did not occur to any of the VFCM.Example 13. Analysis of IL - 12 activity .

The assay for the activity of IL-12 was performed essentially as described in Example 9, wherein SepGI-Null, SepGI-201 , SepGI-207, SepGI-212, SepGI-214, SepGI VFCM of -216 and SepG1-218 infected cells. Cell-based assays were used in which cells with a heterodimeric IL-12 receptor and engineered to have a luciferase gene under the control of an IL-12-responsive promoter (iLite® IL-12 Assay Ready Cells (Eagle Biosciences (Amherst, NH)) were incubated with lysates of cells infected with recombinant HSV. Detection was performed using the One-Glo luciferase system from Promega Corporations.

Briefly, assays were performed by adding diluted VFCM from uninfected cells or cells infected with various HSVs into wells of 96-well plates. Lysates of infected cell cultures (VFCM) were generated as described in Example 2. Serial dilutions of recombinant IL-12 (R&D Systems) were added to additional wells to generate a standard curve. IL-12 reporter cells were basically used according to the manufacturer's instructions. 40K iLite cells were thawed, diluted, and 40 μl was added to each well of a 96-well plate. 40 μl of serial dilutions of VFCM were then added to the assay wells, the contents of the wells were mixed, and the plates were incubated for five hours at 37°C, 5% CO ₂ . Serial dilutions of recombinant IL-12 were added to separate wells to generate a standard curve. One-Glo Luciferase Reagent (Promega Corp., Madison, WI) was then added to each well (40 µL), and after 10 minutes at room temperature, firefly luciferase was measured using a Tecan Spark plate reader of light. The results are shown in Figure 14 , which provides the use of uninfected cell conditioned media, cell conditioned media infected with a virus that does not include the exogenous transgene (SepGI-Null), and infected with viruses containing the IL-12 gene SepGI-201 and Cells of SepGI-207 (no IL-12 gene), SepGI-212 and SepGI-214 (trigene virus with IL-12 gene) and SepGI-216 and SepGI-216 (bigene virus with IL-12 gene) Luminescence diagram of assay of conditioned media. It should be noted that all cells infected with viruses containing IL-12 genes expressed functional IL-12, except for the isolate of the three-gene virus SepGI-214, which also showed no production of IL-12 protein in ELISA (Example 12 ). Example 14. Cell - cell interaction assay .

To assess the ability of the αROR1-αCD3 bispecific antibody encoded by the engineered HSV to bind to target tumor cells and T cells expressing ROR1, mouse tumor cells and human T cells were labeled with a fluorophore, respectively. Hepa 1-6 cells and A549 cells, both expressing ROR1 ( FIG . 15B ), were labeled with eFluor 450 (ThermoFisher), and human T cells isolated from PBMCs were pre-labeled with eFluor 670 ( FIG . 15C ), essentially as in the example Cell-cell interactions were assayed and analyzed by flow cytometry as described in 5. Figure 15D shows an example of flow cytometry results, where bound cells (fluorescing at two wavelengths) can be seen in the upper right region of the figure.

The results of flow cytometry analysis of αROR1-αCD3 bispecific antibody-mediated binding of T cells to ROR1-expressing tumor cells are graphically shown in Figure 16A , Figure 16B and Figure 16C . Figure 16A shows, from left to right, the percentages of Hepa 1-6 cells, A549 wild-type cells, and A549 ROR1 knockout cells that bound to T cells after co-cultivation in the presence of SepGI-218 VFCM expressing the αRSV-αCD3 doublet Constructs of specific antibodies and IL-12. Figure 16B shows, from left to right, the percentage of Hepa 1-6 cells followed by A549 wild-type cells after co-cultivation in the presence of SepGI-201 VFCM expressing a construct encoding the αROR1-αCD3 bispecific antibody. Figure 16C shows, from left to right, the percentage of Hepa 1-6 cells followed by A549 wild-type cells after co-cultivation in the presence of SepGI-216 VFCM expressing bispecific antibodies encoding αROR1-αCD3 and IL-12 The structure. Minimal cell-cell interactions were observed in the presence of SROR1+. In the presence of VFCM between SepGI-201-infected cells and SepGI-216-infected cells, tumor cell-T cell interactions were observed, but not between SepGI-201-infected cells and SepGI-216-infected cells significant difference. Example 15. T cell activation assay .

To determine the effect of the αROR1-αCD3 bispecific antibody on T cell activation, an assay was performed in which tumor cells expressing ROR1 were incubated with T cells in the presence of VFCM of virus-infected cells encoding the αROR1-αCD3 bispecific antibody , followed by assessment of activation markers on the T cell surface. Briefly, wild-type A549 cells, or as a control, ROR1 knockout A549 cells, were plated in wells of a 96-well plate at ¹⁰⁴ cells per well. The next day, freshly isolated CD3+ T cells stained with CFSE were added to the wells at an E:T ratio of 10:1 or 5:1. VFCM was added to the wells as a 1,000-fold dilution, or as a positive control, CD3/CD28 beads were added to the wells (beads:cell ratio 1:20). After one, two and three days, supernatants were removed for staining of T cells for expression of activation markers and analyzed by flow cytometry.

Figures 17A - 17D provide the results of an assay targeting ROR1 knockout A549 cells. Figure 17A shows the relationship between VFCM of uninfected cultures (first set of two columns) or with infected SepGI-Null virus (second set of two columns), SepGI-207 virus (second set of two columns) , SepGI-201 virus (third set of two columns) or CD3/CD28 beads (fourth set of two columns) cells cultured in VFCM irrespective of the cells, on the first, second and third days of the assay, CD3+ The survival rate of T cells is close to 100%. Figure 17B provides CD3+CD4+ cell counts for each assay group on consecutive days of the assay. Figure 17C provides the percentage of CD25+ cells for each analysis group on consecutive days of the assay based on flow cytometry, and Figure 17D provides the percentage of CD69+ cells for each analysis group on consecutive days of the assay. While CD3/CD38 beads elicit T cell activation, as evidenced by increased expression of both CD25 and CD69 during the assay, when ROR1 knockout cells are targeted, regardless of the presence of VFCM, as evidenced by the expression of CD25 and CD69 Assessed, no T cell activation was observed.

Figures 17E - 17H provide the results of an assay targeting wild - type A549 cells expressing ROR1. Figure 17E shows the relationship between VFCM of uninfected cultures (first set of two columns) or with infected SepGI-Null virus (second set of two columns), SepGI-207 virus (second set of two columns) , SepGI-201 virus (third set of two columns) or CD3/CD28 beads (fourth set of two columns) cells cultured in VFCM irrespective of the cells, on the first, second and third days of the assay, CD3+ The survival rate of T cells is close to 100%. Figure 17F provides CD3+CD4+ cell counts for each assay group on consecutive days of the assay. Figure 17G provides the percentage of CD25+ cells for each analysis group on consecutive days of the assay based on flow cytometry, and Figure 17H provides the percentage of CD69+ cells for each analysis group on consecutive days of the assay. It should be noted that the presence of VFCM in cultures infected with SepGI-201 virus, which was engineered to express the αROR1-αCD3 bispecific antibody, could cause T cells in co-cultures to express both CD25 and CD69. No manifestation of this induction was observed in co-cultures that instead included SepGI-207-infected cells, VFCMs with SepGI-207-infected cells, or VFCMs with uninfected cells. Thus, using target cells expressing ROR1, the activation of T cells in co-cultures can be attributed to the presence of the αROR1-αCD3 bispecific antibody, which can engage T cells, causing their activation. Example 16. T cell proliferation / activation assay .

Additional cell culture assays were performed with viruses expressing single and double genes. In these assays, A549 wild-type or A549 ROR1 knockout cells were plated in wells of 96-well plates at ¹⁰⁴ cells per well. Purified human T cells stained with celltrace violet (CTV) dye were added to the wells at effector:target ratios of 10:1 and 5:1, and a 1:1,000 dilution of VFCM was added to the wells. VFCM has SepGI-207 (αRSV-αCD3 bispecific antibody gene), SepGI-201 (αROR1-αCD3 bispecific antibody gene), SepGI-216 (αROR1-αCD3 bispecific antibody gene plus IL-12 gene) and Cells infected with SepGI-218 (αROR1-αCD3 bispecific antibody gene plus IL-12 gene). Plates were incubated for 3 days and flow cytometry was performed after 1, 2 or 3 days to determine cell proliferation by the percentage of CTV+ T cells. Figure 18 shows the results of the assay at an effector:target ratio of 5:1, where only in the case of ROR+ target cells and only in cultures including SepGI-201 and SepGI-216 infected cultures containing αROR1 -Specific T cell proliferation was observed in VFCM of αCD3 bispecific antibody. Example 17. Luciferase - based killing assay using VFCM infecting cells engineered to express αROR1 - αCD3 bispecific antibodies with monogenic, bigenic and trigenic HSV .

An assay was performed to assess the effect of VFCM infection of cells infected with HSV engineered to express an αROR1-αCD3 bispecific antibody on T cell killing of ROR1+ tumor cells. In these assays, target cells (A549 wild-type cells or A549 ROR1 knockout cells as controls) were labeled by transducing the cells with retroviruses to express GFP and firefly luciferase. Luciferase-expressing target cells were plated at ¹⁰⁴ cells per well in 100 µl RPMI-1640+10% FCS in 96-well plates and incubated at 37°C for two days. Freshly isolated human T cells, freshly isolated from PBMCs, were then added to the wells at a ratio of 0.5:1, and a 1,000 or 1:8,000 dilution of VFCM was added to each assay well. The plates were incubated at 37°C for four days, then incubated for 5 minutes in the dark by adding 80 µl Bio-Glo Luciferase Assay Reagent (Promega), and the luminescence was read with a TECAN device (integration time, 500 ms) to assess the number of cells expressing luciferase. Figure 19A shows that in the absence of T cells (effectors), the number of A549 wild-type cells was about ¹⁰⁷ , regardless of the presence or absence of VFCM in the medium or the type of VFCM added. However, in the presence of T cells, A549 wild-type target cells in medium containing VFCM of viruses engineered to express αROR1-αCD3 bispecific antibodies (SepGI-201, SepGI-212 and SepGI-216) The reduction is evident (see Table 2 ). In medium containing VFCM of SepGI-207, SepGI-214 and SepGI218 viruses not engineered to express the αROR1-αCD3 bispecific antibody, no killing of target cells was observed, which is also seen in the graph shown in Figure 19C A plot of the percentage kill is provided. Figures 19B and 19D provide the results when A549 target cells were knocked out using ROR1, showing that T cell effectors do not kill cells that do not express ROR1 regardless of the VFCM (or bispecific antibody) in the co-culture. Example 18. Xcelligence killing assay using 549 WT cells and single, double and triple gene expression ( VFCM ) .

Killing assays were also performed using the xCELLigence® Instant Cell Analyzer (Acea Biosciences, San Diego, CA). In these experiments, A549 wild-type and A549 ROR1 knockout cells were seeded into wells of 96-well E-plates (Acea Biosciences) at 10,000 cells/well in 50 µl RPMI-1640+10% FCS. T cells were added at an effector to target ratio of 0.5:1 and a 1:1,000 dilution of VFCM was added to cell cultures infected with HSV SepGI-Null, SepGI-207, SepGI-212, SepGI-216, and SepGI-218 things. Read the disk continuously for three days. Figure 20 shows the analysis of VFCM including HSV that does not include the αROR1-αCD3 bispecific antibody construct: SepGI-207, SepGI-214, SepGI-218 and SepGI-123 (IL-12 gene only), the degree of proliferation and The pattern was essentially the same as for cultures with no VFCM at all. On the other hand, assays of VFCMs including HSVs engineered to express the αROR1-αCD3 bispecific antibody construct: SepGI-201, SepGI-212 and SepGI-216 showed reduced proliferation, indicating that ROR1+ in these media Target cells are killed. Figure 21 shows the results of a parallel assay in which the target cells were ROR1 knockout cells. In this case, no reduction in proliferation was observed. Example 19. In vivo study of antitumor activity of SepGI - 201 VFCM in NOD / Scid pseudohumanized mouse model .

A study was designed to evaluate the effect of treating tumor-bearing NOD/Scid pseudohumanized mice with VFCM of cells infected with SepGI-201 HSV engineered to express an αROR1-αCD3 bispecific antibody. As a control, some tumor-bearing mice were treated with VFCM of cells infected with SepGI-207 HSV engineered to express the αRSV-αCD3 bispecific antibody. Six groups of eight mice were established using the treatment regimen shown in Table 3 . Table 3. Groups of mice used for VFCM treatment studies . Group tumor cells PBMC VFCM 1 4 × 10 ⁶ A549 wt 4 × 10 ⁶ hPBMCs none 2 4 × 10 ⁶ A549 wt 4 × 10 ⁶ hPBMCs αRSV-αCD3 (SepGI-207) 3 4 × 10 ⁶ A549 wt 4 × 10 ⁶ hPBMCs αROR1-αCD3 (SepGI-201) 4 4 x 10 ⁶ A549 ROR1 KO 4 × 10 ⁶ hPBMCs none 5 4 x 10 ⁶ A549 ROR1 KO 4 × 10 ⁶ hPBMCs αRSV-αCD3 (SepGI-207) 6 4 x 10 ⁶ A549 ROR1 KO 4 × 10 ⁶ hPBMCs αROR1-αCD3 (SepGI-201)

A549 wild-type or A549 ROR1 knockout tumor cells were co-injected subcutaneously with human PBMCs into all mice. Four weeks later, the treatment was started, and 50 µl of VFCM was injected around the tumors of the mice in groups 2-6 every four to five days, for a total of five treatments. Tumor growth and body weight were monitored twice weekly. Tumor volume was measured with calipers, and at approximately the end of the study at week 9, tumor growth inhibition (TGI) was calculated as follows: [1-(relative tumor volume in treatment group)/(relative tumor volume in control group)]× 100. Example 20. In vivo study of antitumor activity of SepGI - 201 VFCM in NSG - B2m KO pseudohumanized mouse model .

A study was designed to evaluate the effect of treating tumor-bearing NSG-B2m knockout pseudohumanized mice with SepGI-201 HSV engineered to express an αROR1-αCD3 bispecific antibody. As a control, groups of mice were treated with SepGI-207 HSV engineered to express an αRSV-αCD3 bispecific antibody. Six groups of eight mice were established using the treatment regimen shown in Table 4 . Table 4. Groups of mice used for VFCM treatment studies . Group tumor cells PBMC Virus 1 5 × 10 ⁶ A549 wt 5 × 10 ⁶ hPBMCs none 2 5 × 10 ⁶ A549 wt 5 × 10 ⁶ hPBMCs SepGI-207 (αRSV-αCD3) 3 5 × 10 ⁶ A549 wt 5 × 10 ⁶ hPBMCs SepGI-201 (αROR1-αCD3) 4 5 x 10 ⁶ A549 ROR1 KO 5 × 10 ⁶ hPBMCs SepGI-207 (αRSV-αCD3) 5 5 x 10 ⁶ A549 ROR1 KO 5 × 10 ⁶ hPBMCs SepGI-201 (αROR1-αCD3)

SEQ ID NO:2 蛋白質 人工 o11抗ROR1抗體重鏈可變域CDR1：

SEQ ID NO:3 蛋白質 人工 o11抗ROR1抗體重鏈可變區CDR2：

SEQ ID NO:4 蛋白質 人工 o11抗ROR1抗體重鏈可變區CDR3：

SEQ ID NO:5 蛋白質 人工 o11抗ROR1抗體ROR1抗體輕鏈可變區

SEQ ID NO:6 蛋白質 人工 o11抗ROR1抗體輕鏈可變區CDR1

SEQ ID NO:7 蛋白質 人工 o11抗ROR1抗體輕鏈可變區CDR2

SEQ ID NO:8 蛋白質 人工 o11抗ROR1抗體輕鏈可變區CDR3

SEQ ID NO:9 蛋白質 人工 o11抗ROR1單鏈抗體(scFv)

SEQ ID NO:10 蛋白質 人工 s10抗ROR1抗體重鏈可變區

SEQ ID NO:11 蛋白質 人工 s10抗ROR1抗體重鏈可變區CDR1

SEQ ID NO:12 蛋白質 人工 s10抗ROR1抗體重鏈可變區CDR2

SEQ ID NO:13 蛋白質 人工 s10抗ROR1抗體重鏈可變區CDR3

SEQ ID NO:14 蛋白質 人工 s10抗ROR1抗體輕鏈可變區

SEQ ID NO:15 蛋白質 人工 s10抗ROR1抗體輕鏈可變區CDR1

SEQ ID NO:16 蛋白質 人工 s10抗ROR1抗體輕鏈可變區CDR2

SEQ ID NO:17 蛋白質 人工 s10抗ROR1抗體輕鏈可變區CDR3

SEQ ID NO:18 蛋白質 人工 s10抗ROR1單鏈抗體(scFv)

SEQ ID NO:19 蛋白質 人工 jlv1011抗ROR1抗體重鏈可變區：

SEQ ID NO:20 蛋白質 人工 jlv1011抗ROR1抗體重鏈可變區CDR1：

SEQ ID NO:21 蛋白質 人工 jlv1011抗ROR1抗體重鏈可變區CDR2：

SEQ ID NO:22 蛋白質 人工 jlv1011抗ROR1抗體重鏈可變區CDR3：

SEQ ID NO:23 蛋白質 人工 jlv1011抗ROR1抗體輕鏈可變區：

SEQ ID NO:24 蛋白質 人工 jlv1011抗ROR1抗體輕鏈可變區CDR1

SEQ ID NO:25 蛋白質 人工 jlv1011抗ROR1抗體輕鏈可變區CDR2

SEQ ID NO:26 蛋白質 人工 jlv1011抗ROR1抗體輕鏈可變區CDR3

SEQ ID NO:27 蛋白質 人工 jlv1011單鏈抗體(scFv)

SEQ ID NO:28 蛋白質 人工 信號肽

SEQ ID NO:29 蛋白質 人工 (G ₄S) ₄連接子

SEQ ID NO:30 蛋白質 人工 替代性GS連接子

SEQ ID NO:31 蛋白質 人工 (G ₄S) ₁連接子

SEQ ID NO:32 蛋白質 人工 Hum291抗CD3抗體重鏈可變區：

SEQ ID NO:33 蛋白質 人工 Hum291抗CD3抗體輕鏈可變區：

SEQ ID NO:34 蛋白質 人工 Hum291抗CD3單鏈抗體(scFv)

SEQ ID NO:35 DNA 人工 編碼具有信號肽-抗ROR1純系O11 scFV-連接子-抗CD3純系hum291 scFV之o11抗ROR1/抗CD3雙特異性抗體前驅體

SEQ ID NO:36 蛋白質 人工 o11抗ROR1/抗CD3雙特異性抗體前驅體： 信號肽 -抗ROR1選殖O11 scFV- 連接子-抗CD3選殖hum291 scFV

SEQ ID NO:37 DNA 人工 編碼具有信號肽 -抗ROR1純系s10 scFV- 連接子-抗CD3純系hum291 scFV之s10抗ROR1/抗CD3雙特異性抗體前驅體

SEQ ID NO:38 蛋白質 人工 s10抗ROR1/抗CD3雙特異性抗體前驅體 信號肽-抗ROR1純系s10 scFV- 連接子-抗CD3純系hum291 scFV

SEQ ID NO:39 DNA 人工 編碼jlv1011抗ROR1/抗CD3雙特異性抗體前驅體具有 信號肽-抗ROR1純系jlv1011 scFV- 連接子-抗CD3純系hum291 scFV

SEQ ID NO:40 蛋白質 人工 jlv1011抗ROR1/抗CD3雙特異性抗體前驅體： 信號肽-抗ROR1純系jlv1011 scFV- 連接子-抗CD3純系hum291 scFV

SEQ ID NO:41 DNA 人工 EF1α/HTLV啟動子

SEQ ID NO:42 DNA 巨細胞病毒 CMV啟動子

SEQ ID NO:43 DNA 人工 編碼呼吸道合胞病毒蛋白質F (RSV) scFV抗體前驅體：信號肽、VH鏈-連接子-VL鏈

SEQ ID NO:44 蛋白質 人工 呼吸道合胞病毒蛋白質F (RSV) scFV抗體前驅體(信號肽-VH鏈-連接子-VL鏈)

SEQ ID NO:45 蛋白質 人工 呼吸道合胞病毒蛋白質F (RSV) scFv抗體：(VH鏈-連接子-VL鏈)

SEQ ID NO:46 DNA 人工 編碼人類IL-12 (p40-2x彈性蛋白-p35)

SEQ ID NO:47 蛋白質 人工 人類IL-12 (p40-2x彈性蛋白-p35)

SEQ ID NO:48 DNA 人工 編碼抗VEGFR-2 VKB8 scFV抗體前驅體(信號肽-VH鏈-連接子-VL鏈-IgG1 Fc區)

SEQ ID NO:49 蛋白質 人工 抗VEGFR-2 VKB8 scFV-Fc1抗體前驅體(信號肽-VH鏈-連接子-VL鏈-IgG1 Fc區)

SEQ ID NO:50 DNA 人工 抗VEGFR-2 VKB8 scFV-Fc1抗體(VH鏈-連接子-VL鏈-IgG1 Fc區)

SEQ ID NO:51 蛋白質 T2A自裂解肽

SEQ ID NO:52 蛋白質 人工 A7抗ROR1抗體重鏈可變區

SEQ ID NO:53 蛋白質 人工 A7抗ROR1抗體重鏈可變區CDR1

SEQ ID NO:54 蛋白質 人工 A7抗ROR1抗體重鏈可變區CDR2

SEQ ID NO:55 蛋白質 人工 A7抗ROR1抗體重鏈可變區CDR3

SEQ ID NO:56 蛋白質 人工 A7抗ROR1抗體輕鏈可變區

SEQ ID NO:57 蛋白質 人工 A7抗ROR1抗體輕鏈可變區CDR1

SEQ ID NO:58 蛋白質 人工 A7抗ROR1抗體輕鏈可變區CDR2

SEQ ID NO:59 蛋白質 人工 A7抗ROR1抗體輕鏈可變區CDR3

SEQ ID NO:60 蛋白質 人工 A8抗ROR1抗體重鏈可變區

SEQ ID NO:61 蛋白質 人工 A8抗ROR1抗體重鏈可變區CDR1

SEQ ID NO:62 蛋白質 人工 A8抗ROR1抗體重鏈可變區CDR2

SEQ ID NO:63 蛋白質 人工 A8抗ROR1抗體重鏈可變區CDR3

SEQ ID NO:64 蛋白質 人工 A8抗ROR1抗體輕鏈可變區

SEQ ID NO:65 蛋白質 人工 A8抗ROR1抗體輕鏈可變區CDR1

SEQ ID NO:66 蛋白質 人工 A8抗ROR1抗體輕鏈可變區CDR2

SEQ ID NO:67 蛋白質 人工 A8抗ROR1抗體輕鏈可變區CDR3

SEQ ID NO:68 蛋白質 人工 2x彈性蛋白連接子

SEQ ID NO:69 蛋白質 人工 (GGGGS)3連接子

A549 wild-type or A549 ROR1 knockout tumor cells were co-injected subcutaneously with human PBMCs into all mice. Four weeks later, the treatment was started, and 50 µl of oncolytic virus was injected around the tumors of the mice in groups 2-5 every four to five days for a total of three treatments. Tumor growth and body weight were monitored twice weekly. Tumor volume was measured with calipers, and at approximately the end of the study at week 9, tumor growth inhibition (TGI) was calculated as follows: [1-(relative tumor volume in treatment group)/(relative tumor volume in control group)]× 100. Sequence SEQ ID NO: 1 protein artificial o11 anti-ROR1 antibody heavy chain variable region

SEQ ID NO: 2 Protein artificial o11 anti-ROR1 antibody heavy chain variable domain CDR1:

SEQ ID NO: 3 Protein artificial o11 anti-ROR1 antibody heavy chain variable region CDR2:

SEQ ID NO: 4 Protein artificial o11 anti-ROR1 antibody heavy chain variable region CDR3:

SEQ ID NO: 5 protein artificial o11 anti-ROR1 antibody ROR1 antibody light chain variable region

SEQ ID NO: 6 protein artificial o11 anti-ROR1 antibody light chain variable region CDR1

SEQ ID NO: 7 protein artificial o11 anti-ROR1 antibody light chain variable region CDR2

SEQ ID NO: 8 protein artificial o11 anti-ROR1 antibody light chain variable region CDR3

SEQ ID NO:9 Protein artificial o11 anti-ROR1 single chain antibody (scFv)

SEQ ID NO: 10 Protein artificial s10 anti-ROR1 antibody heavy chain variable region

SEQ ID NO: 11 Protein artificial s10 anti-ROR1 antibody heavy chain variable region CDR1

SEQ ID NO: 12 Protein artificial s10 anti-ROR1 antibody heavy chain variable region CDR2

SEQ ID NO: 13 Protein artificial s10 anti-ROR1 antibody heavy chain variable region CDR3

SEQ ID NO: 14 protein artificial s10 anti-ROR1 antibody light chain variable region

SEQ ID NO:15 protein artificial s10 anti-ROR1 antibody light chain variable region CDR1

SEQ ID NO: 16 Protein artificial s10 anti-ROR1 antibody light chain variable region CDR2

SEQ ID NO:17 Protein artificial s10 anti-ROR1 antibody light chain variable region CDR3

SEQ ID NO:18 Protein artificial s10 anti-ROR1 single chain antibody (scFv)

SEQ ID NO: 19 Protein artificial jlv1011 anti-ROR1 antibody heavy chain variable region:

SEQ ID NO: 20 Protein artificial jlv1011 anti-ROR1 antibody heavy chain variable region CDR1:

SEQ ID NO: 21 Protein artificial jlv1011 anti-ROR1 antibody heavy chain variable region CDR2:

SEQ ID NO: 22 Protein artificial jlv1011 anti-ROR1 antibody heavy chain variable region CDR3:

SEQ ID NO: 23 protein artificial jlv1011 anti-ROR1 antibody light chain variable region:

SEQ ID NO:24 Protein artificial jlv1011 anti-ROR1 antibody light chain variable region CDR1

SEQ ID NO:25 Protein artificial jlv1011 anti-ROR1 antibody light chain variable region CDR2

SEQ ID NO:26 Protein artificial jlv1011 anti-ROR1 antibody light chain variable region CDR3

SEQ ID NO:27 Protein artificial jlv1011 single-chain antibody (scFv)

SEQ ID NO:28 protein artificial signal peptide

SEQ ID NO:29 Protein artificial (G ₄ S) ₄ linker

SEQ ID NO:30 Protein artificial replacement GS linker

SEQ ID NO:31 Protein artificial (G ₄ S) ₁ linker

SEQ ID NO:32 Protein artificial Hum291 anti-CD3 antibody heavy chain variable region:

SEQ ID NO:33 protein artificial Hum291 anti-CD3 antibody light chain variable region:

SEQ ID NO:34 Protein artificial Hum291 anti-CD3 single chain antibody (scFv)

SEQ ID NO:35 DNA artificially encoded o11 anti-ROR1/anti-CD3 bispecific antibody precursor with signal peptide-anti-ROR1 clonal O11 scFV-linker-anti-CD3 clonal hum291 scFV

SEQ ID NO:36 Protein artificial o11 anti-ROR1/anti-CD3 bispecific antibody precursor: signal peptide - anti-ROR1 colonization O11 scFV- linker -anti-CD3 colonization hum291 scFV

SEQ ID NO:37 DNA artificially encoded s10 anti-ROR1/anti-CD3 bispecific antibody precursor with signal peptide - anti-ROR1 clonal s10 scFV-linker-anti-CD3 clonal hum291 scFV

SEQ ID NO:38 Protein artificial s10 anti-ROR1/anti-CD3 bispecific antibody precursor signal peptide-anti-ROR1 clonal s10 scFV- linker -anti-CD3 clonal hum291 scFV

SEQ ID NO:39 DNA artificially encoded jlv1011 anti-ROR1/anti-CD3 bispecific antibody precursor has signal peptide-anti-ROR1 pure line jlv1011 scFV- linker -anti-CD3 pure line hum291 scFV

SEQ ID NO: 40 Protein artificial jlv1011 anti-ROR1/anti-CD3 bispecific antibody precursor: signal peptide-anti-ROR1 clonal jlv1011 scFV- linker -anti-CD3 clonal hum291 scFV

SEQ ID NO:41 DNA artificial EF1α/HTLV promoter

SEQ ID NO:42 DNA Cytomegalovirus CMV promoter

SEQ ID NO:43 DNA artificially encoded respiratory syncytial virus protein F (RSV) scFV antibody precursor: signal peptide, VH chain-linker-VL chain

SEQ ID NO:44 protein artificial respiratory syncytial virus protein F (RSV) scFV antibody precursor (signal peptide-VH chain-linker-VL chain)

SEQ ID NO:45 Protein artificial respiratory syncytial virus protein F (RSV) scFv antibody: (VH chain-linker-VL chain)

SEQ ID NO:46 DNA artificially encoding human IL-12 (p40-2xelastin-p35)

SEQ ID NO:47 Protein artificial human IL-12 (p40-2xelastin-p35)

SEQ ID NO:48 DNA artificially encoded anti-VEGFR-2 VKB8 scFV antibody precursor (signal peptide-VH chain-linker-VL chain-IgG1 Fc region)

SEQ ID NO:49 protein artificial anti-VEGFR-2 VKB8 scFV-Fc1 antibody precursor (signal peptide-VH chain-linker-VL chain-IgG1 Fc region)

SEQ ID NO:50 DNA artificial anti-VEGFR-2 VKB8 scFV-Fc1 antibody (VH chain-linker-VL chain-IgG1 Fc region)

SEQ ID NO:51 Protein T2A self-cleaving peptide

SEQ ID NO:52 Protein artificial A7 anti-ROR1 antibody heavy chain variable region

SEQ ID NO:53 Protein artificial A7 anti-ROR1 antibody heavy chain variable region CDR1

SEQ ID NO:54 Protein artificial A7 anti-ROR1 antibody heavy chain variable region CDR2

SEQ ID NO:55 Protein artificial A7 anti-ROR1 antibody heavy chain variable region CDR3

SEQ ID NO:56 protein artificial A7 anti-ROR1 antibody light chain variable region

SEQ ID NO:57 protein artificial A7 anti-ROR1 antibody light chain variable region CDR1

SEQ ID NO:58 Protein artificial A7 anti-ROR1 antibody light chain variable region CDR2

SEQ ID NO:59 Protein artificial A7 anti-ROR1 antibody light chain variable region CDR3

SEQ ID NO:60 Protein artificial A8 anti-ROR1 antibody heavy chain variable region

SEQ ID NO:61 Protein artificial A8 anti-ROR1 antibody heavy chain variable region CDR1

SEQ ID NO:62 Protein artificial A8 anti-ROR1 antibody heavy chain variable region CDR2

SEQ ID NO:63 Protein artificial A8 anti-ROR1 antibody heavy chain variable region CDR3

SEQ ID NO:64 protein artificial A8 anti-ROR1 antibody light chain variable region

SEQ ID NO:65 protein artificial A8 anti-ROR1 antibody light chain variable region CDR1

SEQ ID NO:66 protein artificial A8 anti-ROR1 antibody light chain variable region CDR2

SEQ ID NO:67 protein artificial A8 anti-ROR1 antibody light chain variable region CDR3

SEQ ID NO:68 Protein artificial 2x elastin linker

SEQ ID NO:69 Protein artificial (GGGGS) 3 linker

Figure 1 is a schematic diagram showing examples of constructs encoding anti-ROR1/anti-CD3 bispecific antibodies (transcribed from right to left).

Figure 2A illustrates the format of the ELISA detection assay for anti-ROR1/anti-CD3 bispecific antibodies.

Figure 2B provides binding curves of anti-ROR1/anti-CD3 bispecific antibodies produced by cells infected with HSV SepGI-189, SepGI-201 and SepGI-203. Analysis of cell cultures for VFCM.

Figure 3A illustrates the format of a cell binding assay of an anti-ROR1/anti-CD3 bispecific antibody. Wild-type A549 cells express ROR1; ROR1 knockout A549 cells were also tested as controls.

Figure 3B provides the results of a cell binding assay of anti-ROR1/anti-CD3 bispecific antibodies. VFCM of cultures of cells infected with viruses SepGI-189, SepGI-201 and SepGI-203 encoding anti-ROR1/anti-CD3 BspAbs was analyzed.

Figure 4A illustrates the format of the T cell-tumor cell interaction assay

Figure 4B provides the results of a flow cytometric analysis of T cell-tumor cell interactions by the αROR1/αCD3 BsAb present in the VFCM of cultures infected with HSV SepGI-189, SepGI-201 and SepGI-203 mediate.

Figure 5A illustrates the format of a luciferase-based cell signaling assay for an anti-ROR1/anti-CD3 bispecific antibody.

Figure 5B provides the results of a cell signaling assay using VFCM from cultures infected with HSV SepGI-189, SepGI-201 and SepGI-203.

Figure 6 provides percent killing in a cytotoxicity assay involving T cells and VFCM of cultures infected with HSV SepGI-189, SepGI-201 and SepGI-203. The graph on the right also provides interferon gamma (IFNγ) secretion by T cells.

Figure 7A provides a graph of the binding of anti-ROR1 antibodies to A549, A549/ROR1 KO, MCF-7 and HepG2 tumor cells.

Figure 7B provides a graph of percent killing in a cytotoxicity assay using A549, MCF-7, and HepG2 tumor cells as targets, including T cells and cultures infected with SepGI-201 HSV expressing the αROR1/αCD3 BsAb The VFCM. Controls included assays performed in the absence of T cells and assays of VFCM produced from cells infected with SepGI-Null virus that does not express the αROR1/αCD3 BsAb. The results of the IFNγ assay of the co-cultures are also provided.

Figure 8A provides the procedure for the assay of A549 tumor cell killing by HSV expressing the αROR1/αCD3 BsAb.

Figure 8B provides graphs illustrating that virus used to infect cultures enhances killing of ROR1 positive tumor cells and ROR1 knockout cells at various MOIs.

Figure 9A illustrates the format of an ELISA detection assay for the binding of anti-ROR1/anti-CD3 bispecific antibodies to mouse ROR1.

Figure 9B provides the binding curves of antibodies s10 and jlv1011 against ROR1 mice.

Figure 10A provides the tumor inoculation and treatment scheme for an in vivo study of tumor treatment with HSV SepGI-189 and SepGI-201.

Figure 10B provides a graph of tumor volume in A549 tumor inoculated mice treated with HSV SepGI-189 and SepGI-201.

Figure 10C provides a graph of percent tumor growth inhibition in mice treated with HSV SepGI-Null, SepGI-189, and SepGI-201.

Figure 10D provides a graph of body weight during the study shown in Figures 10A, 10B, and 10C.

Figure 11A is a schematic diagram showing an example of a construct encoding an anti-ROR1/anti-CD3 bispecific antibody (transcribed from right to left) and a human IL-12 polypeptide (transcribed from left to right).

Figure 1 IB is a schematic diagram showing examples of constructs encoding anti-ROR1/anti-CD3 bispecific antibodies (transcribed from right to left) and anti-VEGFR2 scFv and human IL-12 polypeptides. Anti-VEGFR2 scFv and human IL-12 polypeptide are transcribed from the same promoter from left to right, and their coding sequences are linked by T2A self-cleaving peptide coding sequence.

Figure 12A provides the results of an ELISA used to detect anti-RSV antibodies in VFCM of cells infected with different HSVs. This figure shows that SepGI-207 and SepGI-218 VFCMs included anti-RSV antibodies.

Figure 12B provides the results of an ELISA used to detect anti-ROR1 antibodies in VFCM of cells infected with different HSVs. This figure shows that SepGI-201, SepGI-212 and SepGI-216 VFCMs included anti-ROR1 antibodies.

Figure 12C provides the results of an ELISA used to detect human IL-12 in VFCM of cells infected with different HSVs. This figure shows that SepGI-212, SepGI-216 and SepGI-218 VFCMs include human IL-12.

Figure 13 provides the results of an ELISA used to detect anti-VEGFR2 antibodies in VFCM of cells infected with different HSVs. This figure shows that the VFCM of isolates of SepGI-212 included anti-VEGFR2 antibodies.

14 is a histogram providing IL in VFCM for detection of cells infected with HSV SepGI-Null, SepGI-201 , SepGI-207, SepGI-212, SepGI-214, SepGI-216 and SepGI-218 Results of assays for -12 activity.

Figure 15A provides the results of flow cytometry of unlabeled tumor cells.

Figure 15B provides the results of flow cytometry of tumor cells labeled with eFluor 450.

Figure 15C provides the results of flow cytometry of human T cells labeled with eFluor 670.

Figure 15D provides the results of flow cytometry of labeled tumor cells and labeled T cells co-incubated with VFCM including anti-ROR1-anti-CD3 bispecific antibody.

Figure 16A provides flow cytometry of tumor cell-T cell interactions mediated by SepGI-218 VFCM (αRSV-αCD3 bsp antibody plus IL-12) when tumor cells are Hepa 1-6, A549 and A549 ROR1 knockout cells Histogram of the results of the measurement assay, expressed as a percentage of analyzed cells.

Figure 16B provides columns of the results of flow cytometric analysis of tumor cell-T cell interactions mediated by SepGI-201 VFCM (αROR1-αCD3 bsp antibody) when the tumor cells were Hepa 1-6 and A549 cells Bar graph, expressed as a percentage of analyzed cells.

Figure 16C provides flow cytometric measurements of tumor cell-T cell interactions mediated by SepGI-216 VFCM (αROR1-αCD3 bsp antibody plus IL-12 and VEGFR2 antibodies) when the tumor cells were Hepa 1-6 and A549 cells A histogram of the results of the assay, expressed as a percentage of analyzed cells.

Figure 17A is a graph showing the percentage of viable CD3+ T cells used in a T cell activation assay over 3 days, where T cells have been incubated in the presence of ROR1 depleted tumor target cells (left to right bars for each day ): VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201 and CD3/CD28 beads. The first column of each pair provides the value of the assay performed at an E:T ratio of 10:1 and the second column of each pair provides the value of the assay performed at an E:T ratio of 5:1 value.

Figure 17B is a graph providing CD3+CD4+ cell counts in each T cell activation assay. The VFCM and E:T ratios of the assay are shown in Figure 17A.

Figure 17C provides CD25+ T cells as a percentage of CD3+CD4+ cells in an activation assay in which T cells were grown in the presence of ROR1+ tumor target cells (bars from left to right for each day): VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201 and CD3/CD28 beads. The first column of each pair provides the value of the assay performed at an E:T ratio of 10:1 and the second column of each pair provides the value of the assay performed at an E:T ratio of 5:1 value.

Figure 17D provides CD69+ T cells as a percentage of CD3+CD4+ cells in an activation assay in which T cells were grown in the presence of ROR1+ tumor target cells (bars from left to right for each day): VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201 and CD3/CD28 beads. The first column of each pair provides the value of the assay performed at an E:T ratio of 10:1 and the second column of each pair provides the value of the assay performed at an E:T ratio of 5:1 value.

Figure 17E is a graph showing the percentage of viable CD3+ T cells used in a T cell activation assay over 3 days, where T cells have been incubated in the presence of ROR1+ tumor target cells (left to right bars for each day) : VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201 and CD3/CD28 beads. The first column of each pair provides the value of the assay performed at an E:T ratio of 10:1 and the second column of each pair provides the value of the assay performed at an E:T ratio of 5:1 value.

Figure 17F is a graph providing CD3+CD4+ cell counts in each T cell activation assay. The VFCM and E:T ratios of the assay are shown in Figure 17E.

Figure 17G provides CD25+ T cells as a percentage of CD3+CD4+ cells in an activation assay in which T cells were grown in the presence of ROR1+ tumor target cells (bars from left to right for each day): VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201 and CD3/CD28 beads. The first column of each pair provides the value of the assay performed at an E:T ratio of 10:1 and the second column of each pair provides the value of the assay performed at an E:T ratio of 5:1 value.

Figure 17H provides CD69+ T cells as a percentage of CD3+CD4+ cells in an activation assay in which T cells were grown in the presence of ROR1+ tumor target cells (bars from left to right for each day): VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201 and CD3/CD28 beads. The first column of each pair provides the value of the assay performed at an E:T ratio of 10:1, and the second column of each pair provides the value of the assay performed at an E:T ratio of 5:1 value.

Figure 18A is a graph showing the percentage of viable CD3+ T cells used in a T cell activation assay over 3 days, where T cells have been incubated in the presence of A549 wild-type cells (ROR1+) tumor target cells (for daily self Left to right bars): VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201, VFCM of cells infected with SepGI-218 and VFCM of cells infected with SepGI-207 216 VFCM of infected cells. The assay was performed with an E:T ratio of 5:1.

Figure 18B is a graph showing the percentage of live CD3+ T cells used in the T cell activation assay over 3 days, where T cells have been incubated in the presence of A549 ROR1 depleted tumor target cells (left to right for each day) Columns): VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepGI-207, VFCM of cells infected with SepGI-201, VFCM of cells infected with SepGI-218, and cells infected with SepGI-216 The VFCM. The assay was performed with an E:T ratio of 5:1.

Figure 19A is a graph showing the results of a luciferase-based toxicity assay in the absence and presence of T cells, where the target was A549 wild-type cells expressing luciferase, and the assay was performed in the presence of uninfected cells Or in the case of VFCM of cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214, SepGI-216 and SepGI-218.

Figure 19B is a graph showing the results of a luciferase-based toxicity assay targeting A549 ROR1 knockout cells expressing luciferase in the absence and presence of T cells and the assay in the presence of uninfected cells Or in the case of VFCM of cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214, SepGI-216 and SepGI-218.

Figure 19C is a graph providing the percent kill for the assay of Figure 17C.

Figure 19D is a graph providing the percent kill for the assay of Figure 17B.

Figure 20 shows the cellular index of cells in an impedance-based cytotoxicity assay using A549 wild-type cells as a target over time. See Example 18.

Figure 21 shows the cellular index of cells in an impedance-based cytotoxicity assay using A549 knockout cells as a target over time. See Example 18.

                <![CDATA[<110> SORRENTO THERAPEUTICS, INC.]]> <![CDATA[<120> Oncolytic virus expressing anti-ROR1/anti-CD3 bispecific antibody]] > <![CDATA[<130> 01223-0096-00PCT]]> <![CDATA[<150> US 63/173,205]]> <![CDATA[<151> 2021-04-09]]> <! [CDATA[<160> 69 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 122]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: o11 anti-ROR1 antibody heavy chain Variable region]]> <![CDATA[<400> 1]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Thr Ser Gly Arg Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Ser Trp Tyr Ser Gly Trp Tyr Phe Asp Leu Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Va l Ser Ser 115 120 <![CDATA[<210> 2]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: o11 anti-ROR1 antibody heavy chain variable region CDR1 ]]> <![CDATA[<400> 2]]> Asn Tyr Tyr Met His 1 5 <![CDATA[<210> 3]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213 > Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic: o11 anti-ROR1 antibody heavy chain variable region CDR2]]> <![CDATA[<400> 3] ]> Ile Ile Asn Pro Thr Ser Gly Arg Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <![CDATA[<210> 4]]> <![CDATA[<211> 13]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: o11 anti-ROR1 antibody heavy chain can Variable region CDR3]]> <![CDATA[<400> 4]]> Asp Ser Ser Ser Trp Tyr Ser Gly Trp Tyr Phe Asp Leu 1 5 10 <![CDATA[<210> 5]]> <![CDATA [<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Synthesis: o11 anti-ROR1 antibody ROR1 antibody light chain variable region]]> <![CDATA[<400> 5]]> Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Thr Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile 85 90 95 Ala Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <![CDATA[<210> ]]> 6 <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>] ]> <![CDATA[<223> Synthesis: o11 anti-ROR1 antibody light chain variable region CDR1 ]]> <![CDATA[<400> 6]]> Arg Ala Ser Gln Gly Ile Arg Thr Asp Leu Ala 1 5 10 <![CDATA[<210> 7]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: o11 anti-ROR1 antibody light chain variable region CDR2]]> <![CDATA[<400> 7]]> Ala Ala Ser Ser Leu Gln Ser 1 5 <![CDATA[<210> 8]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: o11 anti-ROR1 antibody light chain variable region CDR3]]> <![CDATA[<400> 8]]> Gln Gln Tyr Tyr Gly Tyr Pro Ile Ala 1 5 <![CDATA[<210> 9]]> <![CDATA[<211> 249]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>] ]> <![CDATA[<223> Synthesis: o11 anti-ROR1 single chain antibody (scFv)]]> <![CDATA[<400> 9]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Thr Ser Gly Arg Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Ser Trp Tyr Ser Gly Trp Tyr Phe Asp Leu Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile 130 135 140 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 145 150 155 160 Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Thr Asp Leu Ala 165 170 175 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 180 185 190 Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile Ala Phe 225 230 235 240 Gly Gln Gly Thr Arg Leu Glu Ile Lys 245 <![CDATA[<210> 10]]> <![CDATA[<211> 122]]> <![CDATA[<212> PRT]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 antibody heavy chain variable region]]> <![CDATA[<400> 10 ]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Arg Ser Ser Tyr Tyr Leu Trp Val Leu Asp Leu Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <![ CDATA[<210> 11]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 antibody heavy chain variable region CDR1]]> <![CDATA[<400> 11]]> Asn Tyr Tyr Met His 1 5 < ![CDATA[<210> 12]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <! [CDATA[<220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 antibody heavy chain variable region CDR2]]> <![CDATA[<400> 12]]> Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <![CDATA[<210> 13]]> <![CDATA[<211> 13]]> <![CDATA[<212> PRT]] > <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 antibody heavy chain variable region CDR3 ]]> <![CDATA[<400> 13]]> Ser Ser Arg Ser Ser Tyr Tyr Leu Trp Val Leu Asp Leu 1 5 10 <![CDATA[<210> 14]]> <![CDATA[<211 > 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis : s10 anti-ROR1 antibody light chain variable region]]> <![CDATA[<400> 14]]> Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Thr Glu 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <![CDATA[<210> 15]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence ]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 antibody light chain variable region CDR1 ]]> <![CDATA[<400> 15]]> Arg Ala Ser Gln Gly Val Ser Thr Glu Ile Ala 1 5 10 <![CDATA[<210> 16]]> <! [CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA [<223> Synthesis: s10 anti-ROR1 antibody light chain variable region CDR2]]> <![CDATA[<400> 16]]> Ala Ala Ser Ser Ser Leu Gln Ser 1 5 <![CDATA[<210> 17] ]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 antibody light chain variable region CDR3]]> <![CDATA[<400> 17]]> Gln Gln Phe Asn Ser Tyr Pro Ile Thr 1 5 <![CDATA [<210> 18]]> <![CDATA[<211> 249]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220>]]> <![CDATA[<223> Synthesis: s10 anti-ROR1 single chain antibody (s]]>cFv) <![CDATA[<400> 18]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Se r Ser Arg Ser Ser Tyr Tyr Leu Trp Val Leu Asp Leu Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile 130 135 140 Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 145 150 155 160 Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Thr Glu Ile Ala 165 170 175 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 180 185 190 Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Ile Thr Phe 225 230 235 24 0 Gly Gln Gly Thr Arg Leu Glu Ile Lys 245 <![CDATA[<210> 19]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> synthesis: jlv1011 anti-ROR1 antibody heavy chain variable region]]> <![CDATA[<400 > 19]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Lys 20 25 30 Tyr Tyr His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Thr Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Arg Tyr Ser Gly Trp Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Thr Thr Val Thr Val Ser Ser 115 120 <![CDATA[<210> 2]]>0 <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]] > <![CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 antibody heavy chain variable region CDR1]]> <![CDATA[<400> 20]]> Ser Lys Ty r Tyr His 1 5 <![CDATA[<210> 21]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 antibody heavy chain variable region CDR2]]> <![CDATA[<400> 21]]> Ile Ile Asn Pro Thr Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <![CDATA[<210> 22]]> <![CDATA[<211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 antibody heavy chain variable region CDR3]]> <![CDATA[<400> 22]]> Asp Ser Ser Arg Tyr Ser Gly Trp Tyr Phe Asp Leu 1 5 10 <![CDATA[<210> 23]]> <![CDATA[<211 > 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> ] ]> Synthesis: jlv1011 anti-ROR1 antibody light chain variable region: <![CDATA[<400> 23]]> Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Thr Glu 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile 85 90 95 Ala Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <![CDATA[<210> 24] ]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 antibody light chain variable region CDR1]]> <![CDATA[<400> 24]]> Arg Ala Ser Gln Gly Val Ser Thr Glu Ile Ala 1 5 10 < ![CDATA[<210> 25]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <! [CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 antibody light chain variable region CDR2]]> <![CDATA[<400> 25]]> Ala Ala Ser Ser Ser Leu Gln Ser 1 5 <![CDATA[<210> 26]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence] ]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 antibody light chain variable region CDR3]]> <![CDATA[<400> 26]]> Gln Gln Tyr Tyr Gly Tyr Pro Ile Ala 1 5 <![CDATA[<210> 27]]> <![CDATA[<211> 248]]> <![CDATA[<212> PRT]]> <![CDATA[ <213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1 single-chain antibody (scFv)]]> <![CDATA[<400> 27 ]]> Gln Val Gln Leu Val Gln Se r Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Lys 20 25 30 Tyr Tyr His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Thr Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Arg Tyr Ser Gly Trp Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln 130 135 140 Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Thr Glu Ile Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala 180 185 190 Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro Glu Asp Phe 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile Ala Phe Gly 225 230 235 240 Gln Gly Thr Arg Leu Glu Ile Lys 245 <![CDATA[<210> 28]]> <![CDATA[<211> 19]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Signal peptide]]> <![CDATA[<400> 28]]> Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser <![CDATA[<210> 29] ]> <![CDATA[<211> 20]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: (G4S)4 linker]]> <![CDATA[<400> 29]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <![CDATA[<210> 30]]> <![CDATA[<211> 17]]> <![CDATA A[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Alternative GS Linker]] > <![CDATA[<400> 30]]> Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly <![CDATA[<210> 31]]> <![CDATA [<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Synthesis: (G4S)1 linker]]> <![CDATA[<400> 31]]> Gly Gly Gly Gly Ser 1 5 <![CDATA[<210> 32]]> <![CDATA[< 211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Hum291 anti-CD3 antibody heavy chain variable region]]> <![CDATA[<400> 32]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 33]]> <![CDATA[<211> 106]]> <![CDATA [<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Hum291 anti-CD3 antibody light chain variable area]]> <![ CDATA[<400> 33]]> 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 Ser Ala Ser Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 34]]> < ![CDATA[<211> 243]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![ CDATA[<223> Synthetic: Hum291 anti-CD3 single-chain antibody (scFv)]]> <![CDATA[<400> 34]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Arg Al a Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Gly Gly Gly Ser Gly Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Trp Ser Ser Asn Pro Thr Phe Gly Gly Gly Thr Lys Val 225 230 235 240 Glu Ile Lys <![CDATA[<210> 35]]> <![CDATA[<211> 1548]]> <! [CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic: Encoding has signal peptide-anti ROR1 clonal O11 scFV linker-o11 anti-ROR1/anti-CD3 bispecific antibody precursor of anti-CD3 clonal hum291 scFv]]> <![CDATA[<400> 35]]> atggaatgga gttgggtttt cttgtttttc cttagtgtca ccacgggagt ccacaagtgg tgcaa 60 gtacaagtgg tgc aagaaacctg gcgctagcgt gaaggtctcc 120 tgtaaagcaa gtggatatac gttcacaaat tattacatgc actgggtccg ccaagctccc 180 ggtcaagggc tggaatggat gggcattata aaccccacgt caggccgaac ctcctatgca 240 caaaaattcc agggtagagt gaccatgacc agggatacgt ccacaagtac agtttacatg 300 gagctttctt cactccggtc tgaagacact gctgtttatt attgcgcccg cgatagctca 360 agctggtact ctggatggta ctttgacctg tggggacagg ggaccaccgt gacagtatct 420 tcaggaggcg gcggttcagg tggcggtgga agcgggggag gaggctccgg aggcggcgga 480 tccgcgattc agatgacgca atccccaagc agcctcagtg caagtgtagg cgaccgcgtt 540 accatcactt gccgagccag tcaaggaata cgaaccgacc tcgcctggta tcagcagaaa 600 cctgggaagg cgcccaaact tcttatttac gccgcgtcct ctctccagag cggagtgccg 660 agtcgatttt caggaagtgg atctgggacc gatttcacac ttacaatttc aagtcttcag 720 cccgaggact tcgcgacgta ttattgccaa caatattatg gctatcctat agcattcgga 780 caaggaacca ggctcgagat taaaggcggg gggggctctc aagttcaact tgttcaatct 840 ggagcagagg taaagaagcc cggcgcgagc gtaaaggtct catgtaaagc ctcaggttat 900 acattcattt cctacacaat gcactgggtc cggcaggcac ccggtcaagg tctcgaatgg 960 ataggatata tcaatcctcg cagtggctat actcactata accagaagct caaggatcga 1020 gccacgttga ctgcagataa gtctgcaagt accgcatata tggaactttc ctccctccgc 1080 tcagaggaca ctgcagtgta ctactgtgca cggtcagcat attacgatta tgacggattc 1140 gcctactggg gacaaggtac actggtcacc gtaagtagtg gtggcggtag tggtggtgga 1200 agcggtgggg gttccggagg cggttcaggt gacatccaaa tgactcagag cccaagctca 1260 ctttccgcct cagtagggga tcgcgttaca ataacgtgca gtgcctcctc atccgtgagc 1320 tatatgaact ggtaccaaca gaaacctggt aaagctccga agcgcttgat atatgacacg 1380 tcaaagct gg ctagtggagt acccagtagg tttagtggga gcgggagcgg tacagatttc 1440 actctgacaa tatcatcact gcaacctgag gactttgcta cctactattg ccagcaatgg 1500 agtagtaatc cgccgacgtt tggtggggga acgaaggtgg agatcaaa 1548 <![CDATA[<210> 36]]> <![CDATA[<211> 516]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: o11 anti-ROR1/anti-CD3 bispecific antibody Precursor: signal peptide-anti-ROR1 clone O11 scFv linker-anti-CD3 clone hum291 scFv]]> <![CDATA[<400> 36]]> Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gly Gly Gly Leu 50 55 60 Glu Trp Met Gly Ile Ile Asn Pro Thr Ser Gly Arg Thr Ser Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Ser Ser Trp Tyr Se r Gly Trp Tyr Phe 115 120 125 Asp Leu Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Ser Leu Ser Ala Ser Val 165 170 175 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Thr 180 185 190 Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 195 200 205 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 225 230 235 240 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro 245 250 255 Ile Ala Phe Gly Gln Gly Thr Arg Le u Glu Ile Lys Gly Gly Gly Gly 260 265 270 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 275 280 285 Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser 290 295 300 Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 305 310 315 320 Ile Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys 325 330 335 Leu Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala 340 345 350 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly 370 375 380 Gln Val Gly Thr Leu Thr Val Ser Ser Gly Gly Gly Ser Gly Gly Gly 385 390 395 400 Ser Gly Gly Gly Ser Gl y Gly Gly Ser Gly Asp Ile Gln Met Thr Gln 405 410 415 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 420 425 430 Cys Ser Ala Ser Ser Ser Ser Val Ser Val Tyr Met Asn Trp Tyr Gln Gln Lys 435 440 445 Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala 450 455 460 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 465 470 475 480 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 485 490 495 Cys Gln Gln Trp Ser Ser Asn Pro Thr Phe Gly Gly Gly Thr Lys 500 505 510 Val Glu Ile Lys 515 <![CDATA[<210> 37]]> <![CDATA [<211> 1548]]> <![CDATA[<212> D]]>NA <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> Synthesis: encoding s10 anti-ROR1/anti-CD3 bispecific antibody precursor with signal peptide-anti-ROR1 clone s10 scFV linker-anti-CD3 clone hum291 scFv]]> <![CDATA[<400> 37]] > atggaatgg t cctgggtgtt cctgttcttc ctgagcgtga ccacaggcgt gcactctcag 60 gttcagctgg ttcagtctgg cgccgaagtg aagaaacctg gcgcctctgt gaaggtgtcc 120 tgcaaggcca gcggctacac ctttaccaac tactacatgc actgggtccg acaggcccct 180 ggacaaggac ttgagtggat gggcatcatc aaccctagcg gcggcagcac aagctacgcc 240 cagaaattcc agggcagagt gaccatgacc agagacacca gcacctccac cgtgtacatg 300 gaactgagca gcctgagaag cgaggacacc gccgtgtact actgcgccag aagcagcaga 360 tccagctact acctgtgggt gctcgatctg tggggccagg gaacaaccgt gacagtctct 420 tctggtggcg gaggatctgg cggaggtgga agcggcggag gcggtagcgg aggtggtgga 480 tctgcaattc agctgacaca gagccccagc agcctgtctg cctctgtggg agacagagtg 540 acaatcacct gtagagccag ccagggcgtg tccacagaga tcgcttggta tcagcagaag 600 cccggcaagg cccctaagct gctgatctat gctgcctcca gtctgcagag cggcgtgcca 660 tctagatttt ctggcagcgg ctccggcacc gacttcaccc tgacaatatc tagcctgcag 720 ccagaggact tcgccaccta ctactgccag cagttcaaca gctaccccat caccttcggc 780 cagggcacca gactggaaat caaaggtggt ggtggcagcc aggtgcagct cgttcaaagc 840 ggagctgaag tgaaaaagcc aggggcca gc gtgaaagtgt cttgcaaagc ctctggctac 900 acattcatca gctacaccat gcattgggtt cgccaggctc caggccaggg actcgaatgg 960 atcggctaca tcaatcccag aagcggctat acccactaca accagaagct gaaggaccgg 1020 gccacactga ccgccgataa gtctgccagc accgcctata tggaactgtc ctctctgcgg 1080 agcgaagata cagccgtgta ttattgtgcc cgcagcgcct actacgacta cgacggcttt 1140 gcctattggg gacagggcac cctggtcacc gtttcttctg gcggaggaag tggcggcgga 1200 agcggtggtg gttctggcgg tggtagtggc gacatccaga tgacccagtc tccaagctct 1260 ctgagcgcca gcgtgggcga tagagtcacc atcacatgta gcgcctccag cagcgtgtcc 1320 tacatgaact ggtatcaaca aaagcctggg aaagctccca agcgcctgat ctacgacaca 1380 agcaaactgg ccagcggagt gcccagcaga ttttccggat ctggcagtgg cacagacttt 1440 acactcacca taagctcact gcagcccgaa gattttgcca cgtactattg tcagcaatgg 1500 tccagcaatc ctcctacctt cggaggcggc accaaggtcg agatcaag 1548 <![CDATA[<210> 38]]> <![CDATA[<211 > 516]]> <![CDATA[<212> PRT]]> <![CDATA[<213> ]]> Artificial Sequence<![CDATA[<220>]]> <![CDATA[<223> Synthetic : s10 anti-ROR1/anti-CD3]]> bispecific antibody precursor signal peptide-anti-ROR1 clone s10 scFv linker-anti-CD3 clone hum291 scFv <! [CDATA[<400> 38]]> Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Ser Arg Ser Ser Tyr Tyr Leu Trp Val Leu 115 120 125 Asp Leu Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 165 170 175 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Thr 180 185 190 Glu Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 195 200 205 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 225 230 235 240 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro 245 250 255 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly 260 265 270 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 275 280 285 Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser 290 295 300 Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 305 310 315 320 Ile Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys 325 330 335 Leu Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala 340 345 350 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly 370 375 380 Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly Ser Gly Gly Gly 385 390 395 ly 400 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Asp Ile Gln Met Thr Gln 405 410 415 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 420 425 430 Cys Ser Ala Ser Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys 435 440 445 Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala 450 455 460 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 465 470 475 480 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 485 490 495 Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys 500 505 510 Val Glu Ile Lys 515 <![CDATA[<210> 39]]> <![CDATA[<211> 1545]]> <![CDATA[<212> DNA]] > <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Encoding has signal peptide-anti-ROR1 clone jlv1011 scFV linker-anti-CD3純系hum291 scFv之jlv1011抗ROR1/抗CD3雙特異性抗體前驅體]]> <![CDATA[<400> 39]]> atggaatggt cctgggtgtt cctgttcttc ctgagcgtga ccacaggcgt gcactctcag 60 gttcagctgg ttcagtctgg cgccgaagtg aagaaacctg gcgcctctgt gaaggtgtcc 120 tgcaaggcca gcggctacac ctttaccagc aagtactacc actgggtccg acaggcccct 180 ggacaaggac ttgagtggat gggcatcatc aacccccacca gcggcagcac aagctacgcc 240 cagaaattcc agggcagagt gaccatgacc agagacacca gcacctccac cgtgtacatg 300 gaactgagca gcctgagaag cgaggacacc gccg tgtact actgcgccag agacagctct 360 agatacagcg gctggtactt cgacctgtgg ggccagggaa caaccgtgac agtttcttct 420 ggcggcggag gatctggcgg aggtggaagc ggaggcggag gaagcggtgg cggcggatct 480 gctattcagc tgacacagag ccctagcagc ctgtctgcct ctgtgggcga cagagtgaca 540 atcacctgta gagcctctca gggcgtgtcc acagagatcg cctggtatca gcagaagcct 600 ggcaaggccc ctaagctgct gatctatgcc gctagctctc tgcagtccgg cgtgccatct 660 agattttccg gctctggcag cggcaccgac ttcaccctga ccatatctag cctgcagcca 720 gaggacttcg ccacctacta ctgtcagcag tactacggct accctatcgc cttcggccag 780 ggcaccagac tggaaatcaa aggtggcggt ggcagccagg tgcagctcgt tcaaagcgga 840 gctgaagtga aaaagccagg ggccagcgtg aaagtgtctt gcaaagcctc tggctacaca 900 ttcatcagct acaccatgca ttgggttcgc caggctccag gccagggact cgaatggatc 960 ggctacatca atcccagaag cggctatacc cactacaacc agaagctgaa ggaccgggcc 1020 acactgaccg ccgataagtc tgccagcacc gcctatatgg aactgtcctc tctgcggagc 1080 gaagatacag ccgtgtatta ttgtgcccgc agcgcctact acgactacga cggctttgcc 1140 tattggggac agggcaccct ggtcaccgtt tcttctggcg gaggaagtgg cggcggaagc 1200 ggtggtggtt ctggcggtgg tagtggcgac atccagatga cccagtctcc aagctctctg 1260 agcgccagcg tgggcgatag agtcaccatc acatgtagcg cctccagcag cgtgtcctac 1320 atgaactggt atcaacaaaa gcctgggaaa gctcccaagc gcctgatcta cgacacaagc 1380 aaactggcca gcggagtgcc cagcagattt tccggatctg gcagtggcac agactttaca 1440 ctcaccataa gctcactgca gcccgaagat tttgccacgt actattgtca gcaatggtcc 1500 agcaatcctc ctaccttcgg aggcggcacc aaggtcgaga tcaag 1545 <![CDATA[<210 > 40]]> <![CDATA[<211> 515]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthesis: jlv1011 anti-ROR1/anti-CD3 bispecific antibody precursor: signal peptide-anti-ROR1 clone jlv1011 scFV linker-anti-CD3 clone hum291 scFv]]> <![ CDATA[<400> 40]]> Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Lys Tyr Tyr His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Ile Ile Asn Pro Thr Ser Gly Ser Thr Ser Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Ser Ser Arg Tyr Ser Gly Trp Tyr Phe Asp 115 120 125 Leu Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Ser Thr Glu 180 185 190 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200 205 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 210 215 220 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 225 230 235 240 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Tyr Pro Ile 245 250 255 Ala Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly Ser 260 265 270 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 275 280 285 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 290 295 300 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 305 310 315 320 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 325 330 335 Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr 340 345 350 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 355 360 365 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 370 375 380 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Ser 385 390 395 ly 400 Gly Gly Gly Ser Gly Gly Gly Ser Gly Asp Ile Gln Met Thr Gln Ser 405 410 415 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 420 425 430 Ser Ala Ser Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 435 440 445 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser 450 455 460 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 465 470 475 480 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 485 490 495 Gln Gln Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val 500 505 510 Glu Ile Lys 515 <![CDATA[<210> 41]]> <![CDATA[<211> 525]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: EF1-α/HTLV promoter]]> <![CDATA[< 400> 41]]> aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60 cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300 ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360 tgcgtccgcc gtctaggtaa gtttaaagct ca ggtcgaga ccgggccttt gtccggcgct 420 cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480 actctacgtc tttgtttcgt tttctgttct gcgccgttac agatc 525 <![CDATA[<210> 42]]> <![CDATA[<211> 603]]> <![CDATA[<212> DNA ]]> <![CDATA[<213> CMV]]> <![CDATA[<220>]]> <![CDATA[<221> misc_feature]]> <![CDATA[<223> CMV start子]]> <![CDATA[<400> 42]]> aagcttggga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 60 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 120 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 180 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540 agatcgcctg gagacgccat ccacgc tgtt ttgacctcca tagaagacac cgactctact 600 aga 603 <![CDATA[<210> 43]]> <![CDATA[<211> 780]]> <![CDATA[<212> DNA]]> <![CDATA[<213 > Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: encoding respiratory syncytial virus protein F (RSV) scFV antibody precursor: signal peptide, VH chain, linker 、VL鏈]]> <![CDATA[<400> 43]]> atggaatggt cctgggtgtt cctgttcttc ctgagcgtga ccacaggcgt gcacagccaa 60 gtgacactga gagagtctgg ccccgctctg gtcaagccta cacagaccct gacactgacc 120 tgcaccttca gcggctttag cctgagcaca agcggcatga gcgtcggctg gattagacag 180 cctcctggca aagccctgga atggctggcc gacatttggt gggacgacaa gaaggactac 240 aaccccagcc tgaagtcccg gctgaccatc agcaaggaca ccagcaagaa ccaggtggtg 300 ctgaaagtga ccaacatgga ccctgccgac accgccacct actactgtgc cagatccatg 360 atcaccaact ggtacttcga cgtgtgggga gccggcacca cagtgacagt ttctagcgga 420 ggcggaggat ctggtggcgg aggaagtggc ggaggcggtt ctgatatcca gatgacacag 480 agccccagca cactgtctgc cagcgtggga gacagagtga ccatcacatg caagtgccag 540 ctgagcgtgg gctacatgca ctggtatcag cagaagcctg gcaaggcccc taagctgctg 600 atctacgaca caagcaagct ggcctctggc gtgcccagca gattttctgg cag cggcagc 660 ggaaccgagt tcaccctgac catctcaagc ctgcagcctg acgacttcgc tacgtactac 720 tgcttccaag gcagcggcta ccccttcaca tttggaggcg gcaccaagct ggaaatcaag 780 <![CDATA[<210> 44]]> <![CDATA[<211> 260]]> <![CDATA[<212> PRT] ]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Respiratory syncytial virus protein F (RSV) scFV antibody precursor ( Signal peptide-VH chain-linker-VL chain)]]> <![CDATA[<400> 44]]> Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys 20 25 30 Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu 35 40 45 Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60 Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 85 90 95 Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala 100 105 110 Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val 115 120 1 25 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Lys Cys Gln Leu Ser Val Gly Tyr Met His Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala 195 200 205 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe 210 215 220 Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr 225 230 235 240 Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys 245 250 255 Leu Glu Ile Lys 260 <![CDATA[<210> 45]]> <![CDATA[<211> 241]]> <![CDATA[<212> PRT]]> <![CDATA [<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: Respiratory syncytial virus protein F (RSV) scFv antibody: (VH chain, linker, VL Chain)]]> <![CDATA[<400> 45]]> Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Cys 145 150 155 160 Gln Leu Ser Val Gly Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln 210 215 220 Gly Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys <! [CDATA[<210> 46]]> <![CDATA[<211> 1605]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![ CDATA[<220>]]> <![CDATA[<223> Synthetic: Encoding Human IL-12 (p40-2xElastin-p35)]]> <![CDATA[<400> 46]]> atgtgccacc agcagctggt catcagctgg tttagcctgg tgtttctggc ctctccactg 60 gtggccatct gggagctgaa gaaagacgta tacgtggtgg aactggactg gtatcccgat 120 gctcctggcg agatggtggt gctgacctgc gatacccctg aggaagatgg catcacctgg 180 actctggacc agtcctggcctgagaggtagg ccctgaccat ccaagtgaaa 240 gagtttggcg acgccggcca gtacacctgt cacaaaggcg gagaagtgct gagccacagc 300 ctgctgctgc tccacaagaa agaggacggc atctggtcca ccgacatcct gaaggaccag 360 aaagagccta agaacaagac cttcctgcgc tgcgaggcca agaactacag cggcagattc 420 acctgttggt ggctgaccac aatcagcacc gacctgacct tctccgtgaa gtctagcagg 480 ggcagcagtg atcctcaggg cgttacatgt ggcgccgcta cactgtctgc cgaaagagtg 540 cggggcgaca acaaagaata cgagtacagc gtggaatgcc aagaggacag cgcctgtcca 600 gccgccgaag agtctctgcc tatcgaagtg atggtggacg ccgtgcacaa gctgaagtac 660 gagaactaca cctccagctt tttcatccgg gacatcatca agcccgatcc tccaaagaac 720 ctgcagctca agcccctgaa gaacagcaga caggtggaag tgtcttggga gtaccccgac 780 acctggtcta cccctcactc ctacttcagc ctgacctttt gcgtgcaagt gcagggcaag 840 tccaagcgcg agaaaaagga ccgggtgttc accgataaga ccagcgccac cgtgatctgc 900 cgaaagaacg ccagcatcag cgtcagagcc caggaccggt actacagcag ctcttggagc 960 gaatgggcca gcgtgccatg ttctgtgcct ggcgttggag ttcctggcgt gggcagaaat 1020 ctgccagtgg ccacgcctga tcctggcatg tttccttgtc tgcaccactc ccagaac ctg 1080 ctgagagccg tgtccaatat gctgcagaag gcccggcaga cactggaatt ctacccctgc 1140 accagcgagg aaatcgacca cgaggatatc accaaggaca agaccagcac cgtggaagcc 1200 tgcctgcctc tggaactgac aaagaacgag agctgcctga acagccggga aaccagcttc 1260 atcaccaacg gctcttgcct ggcctccaga aagacctcct tcatgatggc cctgtgcctg 1320 agcagcatct acgaggacct gaagatgtac caggtggaat tcaagaccat gaacgccaag 1380 ctgctgatgg accccaagag acagatcttc ctggaccaga acatgctggc cgtgatcgat 1440 gagctgatgc aggccctgaa cttcaacagc gagacagtgc cccagaagtc cagcctggaa 1500 gaacccgact tctataagac caagatcaag ctgtgcatcc tgctgcacgc cttccggatc 1560 agagccgtga ccatcgacag agtgatgagc tacctgaacg cctcc 1605 <![CDATA[<210> 47]]> <![CDATA[<211> RT 535]]>[<]1>ATA <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic: Human IL-12 (p40-2xElastin-p35)]]> <![CDATA[<400> 47]]> Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 2 00 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser Val Pro Gly Val Gly Val Pro Gly 325 330 335 Val Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro 340 345 350 Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu 355 360 365 Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu 370 375 380 Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala 385 390 395 400 Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg 405 410 415 Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr 420 425 430 Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys 435 440 445 Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp 450 455 460 Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp 465 470 475 480 Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln L ys 485 490 495 Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys 500 505 510 Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val 515 520 525 Met Ser Tyr Leu Asn Ala Ser 530 535 <![CDATA[<210> 48]]> <![CDATA[<211> 1491]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: encoding anti-VEGFR-2 VKB8 scFV antibody precursor (signal peptide-VH chain-linker-VL chain-IgG1 Fc domain)]] > <![ CDATA[<400> 48]]> atggaatggt cctgggtgtt cctgttcttc ctgagcgtga ccacaggcgt gcactctgaa 60 gtgcagctgg ttcagtctgg cgccgaagtg aagaaacctg gcagcagcgt gaaggtgtcc 120 tgcaaggctt acggcggcac ctttggctct tatggcgtgt cctgggttcg cagagcacct 180 ggacaaggcc tggaatggat gggcagactg atccccatct tcggcaccag agactacgcc 240 cagaaattcc agggcagagt gaccctgaca gccgacgagt ctaccaacac cgcctacatg 300 gaactgagca gcctgagaag cgaggacacc gccgtgtact actgtgccag agatggcgac 360 tactacggca gcggcagcta ctatggcatg gatgtgtggg gccagggcac cctggttaca 420 gtttcttctg gtggcggagg atctggcgga ggtggaagcg gcggaggcgg atctgaaaca 480 acactgacac agagccccgc cacactgagt gtgtctccag gcgaaagggc caccgtgtct 540 tgtcgagcct ctcagagcct gggcagcaac ctcggatggt tccagcagaa accaggacag 600 gcccctcggc tgctgatcta tggcgcttct acaagagcca caggcatccc cgccagattt 660 tctggctctg gcagcggaac cgagttcacc ctgacaatct ctagcctgca gtccgaggac 720 ttcgctgtgt acttctgcca gcagtacaac gactggccca tcacattcgg ccaggggacc 780 aagctggaaa tcaaagagcc caagagcagc gacaagaccc aacctgtcc tccatgtcct 840 gct cctgaac tgctcggcgg accttccgtg tttctgttcc ctccaaagcc taaggacacc 900 ctgatgatca gcagaacccc tgaagtgacc tgcgtggtgg tggatgtgtc ccacgaggac 960 ccagaagtga agttcaactg gtatgtggac ggcgtggaag tgcacaacgc caagaccaag 1020 cctagagagg aacagtacaa cagcacctac agagtggtgt ccgtgctgac cgtgctgcac 1080 caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggc cctgcctgct 1140 cctatcgaga aaaccatcag caaggccaag ggccagccta gggaacccca ggtttacaca 1200 ctgcctccaa gcagggacga gctgaccaag aatcaggtgt ccctgacctg cctggtcaag 1260 ggcttctacc cttccgatat cgccgtggaa tgggagagca atggccagcc agagaacaac 1320 tacaagacca ctcctcctgt gctggacagc gacggctcat tcttcctgta ctccaagctg 1380 acagtggaca agagcagatg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag 1440 gccctgcaca accactacac acagaagtcc ctgtctctga gccccggcaa g 1491 <![CDATA[<210> 49]]> <![CDATA[<211> 497]] > <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic: Anti-VEGFR- 2 VKB8 scFV-Fc1 antibody precursor (signal peptide-VH chain-linker-VL chain-IgG1 Fc domain)]]> <![CDATA[<400> 49]]> Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Tyr Gly Gly Thr Phe 35 40 45 Gly Ser Tyr Gly Val Ser Trp Val Arg Arg Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Arg Leu Ile Pro Ile Phe Gly Thr Arg Asp Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Asn 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Asp Tyr Tyr Gly Ser Gly Ser Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr 145 150 155 160 Thr Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg 165 170 175 Ala Thr Val Ser Cys Arg Ala S Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly 210 215 220 Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 225 230 235 240 Phe Ala Val Tyr Phe Cys Gln Gln Tyr Asn Asp Trp Pro Ile Thr Phe 245 250 255 Gly Gln Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Ser Asp Lys 260 265 270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 275 280 285 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 305 310 315 320 Pro Glu Val Lys P he Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser Val Leu Thr Val Leu Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 370 375 380 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390 395 400 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly A sn Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys <![CDATA[<210> 50]]> <![CDATA [<211> 478]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Synthesis: anti-VEGFR-2 VKB8 scFV-Fc1 antibody (VH chain-linker-VL chain-IgG1 Fc domain)]]> <![CDATA[<400> 50]]> 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 Tyr Gly Gly Thr Phe Gly Ser Tyr 20 25 30 Gly Val Ser Trp Val Arg Arg Ala Pro Gly Gly Gly Gly Leu Glu Trp Met 35 40 45 Gly Arg Leu Ile Pro Ile Phe Gly Thr Arg Asp Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asp Tyr Tyr Gly Ser Gly Ser Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr Thr Leu Thr 130 135 140 Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Val 145 150 155 160 Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Asn Leu Gly Trp Phe Gln 165 170 175 Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr 180 185 190 Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val 210 215 220 Tyr Phe Cys Gln Gln Tyr Asn Asp Trp Pro Ile Thr Phe Gly Gln Gly 225 230 235 240 Thr Lys Leu Glu Ile L Glu Pro Lys Ser Ser Asp Lys Thr His Thr 245 250 255 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 325 330 335 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345 350 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 355 360 365 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 370 375 380 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <![CDATA[<210> 51]] > <![CDATA[<211> 21]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> < ![CDATA[<223> Synthesis: T2A self-cleaving peptide]]> <![CDATA[<400> 51]]> Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <![CDATA[<210> 52]]> <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence ]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A7 anti-ROR1 antibody heavy chain variable domain]]> <![CDATA[<400> 52]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 1 0 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Ser Ser Ala Tyr Ser Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Leu Leu Tyr Gly Trp Leu Thr Asp Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 53]]> <![CDATA[<211> 5]]> < ![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A7 anti-ROR1 antibody heavy chain CDR1]]> <![CDATA[<400> 53]]> Asp Tyr Tyr Met Thr 1 5 <![CDATA[<210> 54]]> <![CDATA[<211> 17]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A7 anti-ROR1 antibody heavy chain CDR2]]> <![CDATA[<400> 54]]> Tyr Ile Ser Gly Ser Ser Ala Tyr Ser Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[<210> 55]]> < ![CDATA[<211> 10]]> <![CDATA[<212> PRT ]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A7 anti-ROR1 antibody heavy chain CDR3]]> <![ CDATA[<400> 55]]> Asp Pro Leu Leu Tyr Gly Trp Leu Thr Asp 1 5 10 <![CDATA[<210> 56]]> <![CDATA[<211> 102]]> <![CDATA [<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A7 anti-ROR1 antibody light chain variable Domain]]> <![CDATA[<400> 56]]> Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Val Ser Trp Tyr Gln 20 25 30 Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Val Ser Lys 35 40 45 Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn 50 55 60 Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp 65 70 75 80 Tyr Tyr Cys Ser Ser Tyr Ile Asn Asp Ala Val Phe Phe Gly Gly Gly 85 90 95 Thr Lys Leu Thr Val Leu 100 <![CDATA[<210> 57]] > <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> < ![CDATA[<223> Synthesis: A7 anti-ROR1 antibody light chain CDR1]]> <![CDATA[<400> 57]]> Thr Gly Thr Ser Ser 1 5 <![CDATA[<210> 58]]> <![CDATA[< 211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A7 anti-ROR1 antibody light chain CDR2]]> <![CDATA[<400> 58]]> Glu Val Ser Lys Arg Pro Ser 1 5 <![CDATA[<210> 59]]> <![CDATA[ <211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223 > Synthesis: A7 anti-ROR1 antibody light chain CDR3]]> <![CDATA[<400> 59]]> Ser Ser Tyr Ile Asn Asp Ala Val Phe 1 5 <![CDATA[<210> 60]]> <! [CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA [<223> Synthesis: A8 anti-ROR1 antibody light chain variable domain]]> <![CDATA[<400> 60]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Ser Ser Ala Tyr Ser Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Leu Leu Tyr Gl y Trp Leu Thr Asp Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 61]]> <![CDATA[<211> 5]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A8 anti-ROR1 antibody heavy chain CDR1]]> <![CDATA[<400> 61]]> Asp Tyr Tyr Met Thr 1 5 <![CDATA[<210> 62]]> <![CDATA[<211> 17]]> <![CDATA[<212 > PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A8 Anti-ROR1 Antibody Heavy Chain CDR2]]> < ![CDATA[<400> 62]]> Tyr Ile Ser Gly Ser Ser Ala Tyr Ser Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[<210> 63]]> <![CDATA[< 211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A8 anti-ROR1 antibody heavy chain CDR3]]> <![CDATA[<400> 63]]> Asp Pro Leu Leu Tyr Gly Trp Leu Thr Asp 1 5 10 <![CDATA[<210> 64]]> < ![CDATA[<211> 109]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![ CDATA[<223> Synthetic: A8 anti-ROR1 antibody light chain variable domain]]> <![CDATA[<400> 64]]> Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Gly Gly Gly Tyr 20 25 30 Asp Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Asn Lys Arg Pro Ser Gly Val Ser Gly Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Thr Ser Asp 85 90 95 Val Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <![CDATA[<210> 65]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213 > Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthetic: A8 anti-ROR1 antibody light chain CDR1]]> <![CDATA[<400> 65]]> Thr Gly Thr Ser Ser Asp Gly Gly Gly Tyr Asp Ser Val Ser 1 5 10 <![CDATA[<210> 66]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT] ]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A8 anti-ROR1 antibody light chain CDR2]]> <![CDATA [<400> 66]]> Asp Val Asn Lys Arg Pro Ser 1 5 <![CDATA[<210> 67]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT ]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: A8 anti-ROR1 antibody light chain CDR3]]> <![ CDATA[<4 00> 67]]> Ser Ser Phe Thr Ser Asp Val Met Val 1 5 <![CDATA[<210> 68]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT ]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: 2×Elastin Linker]]> <![CDATA [<400> 68]]> Val Pro Gly Val Gly Val Pro Gly Val Gly 1 5 10 <![CDATA[<210> 69]]> <![CDATA[<211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Synthesis: (GGGGS)3 Linker]]> <![CDATA[<400> 69]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
        

Claims (54)

A bispecific antibody comprising a ROR1-binding single-chain variable fragment antibody (ScFv) and a CD3-binding single-chain variable fragment antibody (ScFv), wherein the anti-ROR1 scFv and the anti-CD3 scFv are joined via a linker, and further among them: The ScFv anti-ROR1 antibody has a heavy chain variable domain that is at least 95% identical to SEQ ID NO:1 and a light chain variable domain that is at least 95% identical to SEQ ID NO:5; The anti-ROR1 ScFv has a heavy chain variable domain that is at least 95% identical to SEQ ID NO: 10 and a light chain variable domain that is at least 95% identical to SEQ ID NO: 14; The anti-ROR1 ScFv has a heavy chain variable domain that is at least 95% identical to SEQ ID NO: 19 and a light chain variable domain that is at least 95% identical to SEQ ID NO: 23; The anti-ROR1 ScFv has a heavy chain variable domain at least 95% identical to SEQ ID NO:52 and a light chain variable domain at least 95% identical to SEQ ID NO:56; or The anti-RORI ScFv has a heavy chain variable domain at least 95% identical to SEQ ID NO:60 and a light chain variable domain at least 95% identical to SEQ ID NO:64. The bispecific antibody according to claim 1, wherein the anti-ROR1 scFv has a heavy chain variable domain with at least 95% identity to SEQ ID NO:1 and a light chain with at least 95% identity to SEQ ID NO:5 variable domain. The bispecific antibody according to claim 2, wherein the anti-ROR1 scFv comprises an amino acid sequence having at least 95% identity with SEQ ID NO:9. The bispecific antibody according to claim 1, wherein the anti-ROR1 ScFv has a heavy chain variable domain with at least 95% identity to SEQ ID NO:10 and a light chain with at least 95% identity to SEQ ID NO:14 Variable domain. The bispecific antibody according to claim 4, wherein the anti-ROR1 scFv comprises an amino acid sequence having at least 95% identity to SEQ ID NO:18. The bispecific antibody according to claim 1, wherein the anti-ROR1 ScFv has a heavy chain variable domain with at least 95% identity to SEQ ID NO: 19 and a light chain with at least 95% identity to SEQ ID NO: 23 Variable domain. The bispecific antibody according to claim 6, wherein the anti-ROR1 scFv comprises an amino acid sequence having at least 95% identity with SEQ ID NO:27. The bispecific antibody according to claim 1, wherein the anti-CD3 scFv comprises a heavy chain variable domain with at least 95% identity to SEQ ID NO:32 and a light chain with at least 95% identity to SEQ ID NO:33 Variable domain. The bispecific antibody according to claim 8, wherein the anti-CD3 scFv comprises an amino acid sequence having at least 95% identity to SEQ ID NO:34. The bispecific antibody according to any one of the preceding claims, wherein the heavy chain variable domain and the light chain variable domain of the scFv antibodies are joined via a GS linker. A nucleic acid construct encoding the anti-ROR1/anti-CD3 bispecific antibody according to any one of claims 1 to 10. The nucleic acid construct according to claim 11, wherein the coding sequence of the anti-ROR1/anti-CD3 bispecific antibody includes a sequence coding for a signal peptide at its N-terminus. The nucleic acid construct according to claim 11, wherein the anti-ROR1/anti-CD3 bispecific antibody coding sequence is operably linked to a promoter. The nucleic acid construct as claimed in item 13, wherein the promoter is EF1α promoter, CMV promoter, JET promoter, RSV promoter, SV40 promoter, CAG promoter, β-actin promoter, HTLV promoter or EF1α/HTLV hybrid promoter. The nucleic acid construct according to claim 13, further comprising a polyadenylation sequence linked to the 3' end of the anti-ROR1/anti-CD3 bispecific antibody coding sequence. A recombinant viral genome comprising the nucleic acid construct according to any one of claims 11-15. A recombinant oncolytic virus comprising the nucleic acid construct according to any one of claims 11-15. The recombinant oncolytic virus according to claim 17, wherein the oncolytic virus is herpes simplex virus (HSV). The recombinant oncolytic virus according to claim 18, wherein the oncolytic virus is HSV-1. The recombinant oncolytic HSV according to claim 19, wherein the oncolytic HSV further comprises a nucleic acid sequence encoding IL-12. The recombinant oncolytic HSV according to claim 20, wherein the IL-12 is human IL-12. The recombinant oncolytic HSV according to claim 20, wherein the IL-12 comprises the amino acid sequence of SEQ ID NO:47. The recombinant oncolytic HSV according to claim 19, wherein the oncolytic HSV further comprises a nucleic acid sequence encoding an anti-VEGFR antibody. The recombinant oncolytic HSV according to claim 23, wherein the anti-VEGFR antibody comprises SEQ ID NO:49. The recombinant oncolytic HSV according to claim 19, wherein the oncolytic HSV is derived from HSV-1 strain 17, HSV-1 strain F, HSV-1 strain KOS or HSV-1 strain JS1. The recombinant oncolytic HSV according to claim 25, wherein the oncolytic HSV is derived from HSV strain 17. The recombinant oncolytic HSV according to any one of claims 19 to 26, wherein the oncolytic HSV does not encode a functional ICP34.5 encoding gene. The recombinant oncolytic HSV according to claim 27, wherein all or part of the ICP34.5 coding gene is deleted. The recombinant oncolytic HSV according to claim 27, wherein the nucleic acid construct encoding the anti-ROR1/anti-CD3 bispecific antibody is inserted into the ICP34.5 encoding locus. A recombinant oncolytic virus for use in a method of treating cancer, wherein the method comprises administering the oncolytic virus according to any one of claims 17 to 29 to an individual suffering from cancer. The recombinant oncolytic HSV according to claim 30, wherein the oncolytic virus is oncolytic HSV. The recombinant oncolytic HSV according to claim 31, wherein the method comprises administering the oncolytic HSV by intravenous, intracavitary, intraperitoneal, intratumoral or peritumoral delivery. The recombinant oncolytic HSV according to claim 32, wherein the delivery is via catheter, infusion or injection. The recombinant oncolytic HSV according to any one of claims 30 to 33, wherein the method comprises administering to the individual more than one dose of the oncolytic HSV. The recombinant oncolytic HSV according to any one of claims 30 to 34, wherein the cancer is a solid tumor. The recombinant oncolytic HSV according to any one of claims 30 to 35, wherein the individual is a dog, a horse or a primate. The recombinant oncolytic HSV according to claim 36, wherein the individual is human. A pharmaceutical composition comprising the recombinant oncolytic HSV according to any one of claims 17 to 37 and pharmaceutically acceptable excipients. The pharmaceutical composition according to claim 38, wherein the concentration of the oncolytic HSV is at least 10 ⁶ /ml. The pharmaceutical composition according to claim 39, wherein the concentration of the oncolytic HSV is at least 10 ⁷ /ml. A method of treating cancer in an individual comprising administering an oncolytic HSV or a pharmaceutical composition according to any one of claims 17-40 to an individual suffering from cancer. The method according to claim 41, wherein the individual is a dog, a horse or a primate. The method of claim 42, wherein the individual is human. The method of claim 41, comprising administering the oncolytic HSV by intravenous, intraarterial, intracavity, intratumoral or peritumoral delivery. The method of claim 44, wherein the delivery is by catheter, infusion or injection. The method of any one of claims 41 to 45, comprising administering to the individual more than one dose of the oncolytic HSV. The method according to any one of claims 41 to 46, wherein the cancer is a solid tumor. A host cell infected with the oncolytic virus according to any one of claims 17-29. The host cell according to claim 48, wherein the host cell is Vero cell, HEK293 cell or BHK cell. A method for producing a pharmaceutical virus composition, comprising culturing the host cell as claimed in claim 48 to produce a virus supernatant and isolating viruses from the virus supernatant to produce a pharmaceutical virus composition. A virus-free conditioned medium (VFCM), which comprises the bispecific antibody according to any one of claims 1-10. A method of treating cancer comprising treating an individual with a pharmaceutical composition comprising the anti-ROR1/anti-CD3 bispecific antibody according to any one of claims 1-10. A method of treating cancer comprising treating an individual with a pharmaceutical composition comprising a VFCM according to claim 51. The method of claim 53, wherein the individual is a non-human individual.

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