CA3214641A1

CA3214641A1 - Oncolytic viruses expressing anti-ror1/anti-cd3 bispecific antibodies

Info

Publication number: CA3214641A1
Application number: CA3214641A
Authority: CA
Inventors: Damien Bresson; Colin Powers; Robert Allen
Original assignee: Sorrento Therapeutics Inc
Current assignee: Sorrento Therapeutics Inc
Priority date: 2021-04-09
Filing date: 2022-04-08
Publication date: 2022-10-13
Also published as: TW202305000A; CN117460833A; JP2024513238A; WO2022217048A1; EP4320243A1

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 VIRUSES EXPRESSING

[0001] This application claims the benefit of priority under 35 U.S.C. 119 to U.S.
provisional application No. 63/173,205, filed April 9, 2021, the entire contents of which are incorporated by reference in its entirety.
TECHNICAL FIELD

[0002] The present disclosure provides bispecific antibodies that bind to both ROR1 and CD3 simultaneously. The present disclosure provides anti-ROR1/anti-CD3 bispecific antibodies, nucleic acids encoding the anti-ROR1/anti-CD3 bispecific antibodies, oncolytic viruses that include constructs encoding anti-ROR1/anti-CD3 bispecific antibodies, and methods of use in treating cancer.
BACKGROUND

[0003] Receptor tyrosine kinase orphan receptors-1 and -2 (ROR1 and ROF,.2) have been described as being specifically associated with particular cancers (RehaYay et al., 2012, Froni Oncol., 2(34)), while being largely absent in. expression on healthy tissue with few exceptions (Balakrishnan at al., 2017, (7/in. Cancer Res., 23(12), 3061-3071). Due to the very tumor--selective expression of the ROR lamily members, they represent relevant targets for targeted cancer therapies.

[0004] Receptor tyrosine kinase orphan receptor-1. (ROR1) is aberrantly expressed in B-cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (3,4CL). ROM
exhibits nearly 100% association with chronic lymphocytic leukemia (CLL.) (Cm et al., 2016, Mood, 128(25), 2931) and has been established as a. marker for some acuteNnwhoblastic leukemias (ALL), mantle cell lymphomas, and some other blood malignancies.
ROR1 is also expressed in certain solid tumors, such as those of lung and breast cancer (Ballakrishnan et al., 2017, (7/in. Cancer Res., 23(12), :3061-3071). ROR1 has been found to he involved in progression of a number of solid tumors, such as rieuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer, head and neck cancer, and melanoma and has been shown to inhibit apoptosis, potentiate -EGFR signaling, induce epithelial-ITICS C In al transition t:All), and contribute to CaVCOlae format/

[0005] ROR1 is mainly detectable in embryonic tissue and generally absent in adult tissue, making the protein an ideal drug target for cancer therapy. ROR1 has therefore been recognized as a target for the development of ROR1 specific antibodies.
However, due to the hi2.1-i homology of ROR1 between different mammalian species, which is 100%
conserved on the amino acid level between humans and cynomolgus monkeys, 96.7% homologous between human and mouse, and 96.3% homologous between human and rabbit, it has been difficult to raise high affinity antibodies against this target by standard technologies, such as animal immunizations

[0006] Oncolytic viruses are viruses that selectively infect and lyse cancer cells. Oncolytic viruses have been the subject of clinical trials for the treatment 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 demonstrated the safety of oncolytic herpes simplex viruses (HSVs) attenuated in their ability to replicate in normal cells by deletion of at least one copy of the gene encoding ICP34.5 (Rampling et al. (2000) Gene Therapy

7:859-866;
Papanastassiou etal. (2002) Gene Therapy 9:398-406; Makie et al. (2001) Lancet 357:525-526; Marken etal. (2000) Gene Therapy 7:867-874; Marken etal. (2009) Molecular Therapy 17:199-207; Senzer etal. (2009) J Clin Oncol 27:5763-5771).
[0007] In addition to directly attacking the tumor by lysing cancer cells, oncolytic HSVs can induce an anti-tumor immune response in the patient (Papanastassiou et at.
(2002);
Marken et al. (2009); Senzer et al. (2009)) as viral antigens are expressed on infected cancer cells and tumor antigens are released when cancer cells are lysed. Viruses also engage mediators of the innate immune response as part of the host recognition of -viral infection resulting in an inflammatory response (Hu et al. (2006) Clin Cancer Res.
12:6737-6747).
These immune responses to treatment with oncolytic viruses may provide a systemic benefit to cancer patients resulting in the suppression of tumors which have not been infected with the virus, including metastatic tumors, and may prevent disease rec-urrence.
SUMMARY

[0008] The present application ("ascribes bispecific antibodies that simultaneously bind RORI and CD3 (aROR1/aCD3 Bsp Abs). Constructs encoding aROR1/aCD3 Bsp Abs as described herein were cloned into an oncolytic HSV-1 virus ("Seprehvec") derived from HSV 17 that does not include a functional RL-1 gene. Virus-infected cells were used to produce Virus Free Cell Media (VFCMs) that include bispecific antibodies which were tested for their ability to enhance cytotoxicity of T cells toward ROR1-expressing tumor cells. The aROR 1 /aCD3 BspAbs disclosed herein demonstrated potent T cell targeting with specific anti-tumor activity in preclinical studies. The expression of an aROR1/aCD3 BspAb by the oncolytic Seprehvec HSV significantly increased anti-tumor activity of viral treatment in an antigen-dependent manner.

[0009] Provided herein in a first aspect is a bispecific antibody comprising a first single chain variable fragment antibody (ScFv) that binds ROR1 and a second single chain variable fragment antibody (ScFv) that binds CD3, wherein the anti-RORI scFv and the anti-CD3 scFv are joined via a linker. The 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, for example, from 1-8. The anti-ROR1/anti-CD3 bispecific antibody (aROR1/aCD3 BspAb) can be an isolated protein, and in some examples is partially or substantially purified.

[0010] The anti-ROR1 scFv of the bispecific antibody can have a heavy chain variable domain (VH) sequence and a light chain variable domain (VL) sequence connect by a linker, such as a (G4S)n linker, and the VH and VL sequences can be derived from a monoclonal antibody that binds ROR1, for example, binds the human ROR1 protein. For example, the anti-ROR1 scFv of the bispecific antibody can include 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:1, 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:5. In another example, the anti-ROR1 scFv of the bispecific antibody can include 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:10, 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:14.
In a further example, the anti-ROR1 scFv of the bispecific antibody can include 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:19, 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:23.

[0011] In further examples, the anti-ROR1 scFv of the bispecific antibody can include 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: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 the anti-ROR1 scFv of the bispecific antibody can include 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:60, 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:64.

[0012] In various embodiments, the anti-ROR1 scFv of anti-ROR1/anti-CD3 bispecific antibody (aROR1/aCD3 BspAb) as provided herein can have an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to SEQ ID
NO:9, SEQ ID NO:18, or SEQ ID NO:27.
100131 The anti-CD3 scFv of the aROR1/aCD3 BspAb provided herein can be, in nonlimiting embodiments, an anti-CD3 scFv that comprises a heavy chain variable domain having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to SEQ
ID NO:32 and a light chain variable domain having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:33. In some embodiments the anti-CD3 scFv comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:34.
100141 A further aspect provided herein are nucleic acid constructs encoding any of the aROR1/aCD3 BspAbs disclosed herein. The aROR1/aCD3 BspAb encoded by the nucleic acid construct can 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 that includes a promoter operably linked to the aROR1/aCD3 BspAb encoding sequence. The promoter can be, as nonlimiting examples, an EF1 a promoter, a CMV promoter (e.g., SEQ ID NO:42), a JeT
promoter, an RSV promoter, an SV40 promoter, a CAG promoter, a beta-actin promoter, an HTLV

promoter, or an EFla/HTLV hybrid promoter (e.g., SEQ ID NO:41). The nucleic acid construct can further include a polyadenylation sequence 3' of the BspAb-encoding sequence, such as, for example, an SV40 3' sequence. The nucleic acid construct can be provided in a vector, and in some examples may be cloned into a recombinant viral genome.
100151 A further aspect provided herein is a recombinant oncolytic virus comprising a nucleic acid construct comprising a nucleic acid sequence encoding an aROR1/aCD3 BspAb according to any disclosed herein. In various embodiments the recombinant oncolytic virus is a recombinant herpes simplex virus (HSV), for example, and HSV-1 virus such as a virus derived from HSV-1 strain 17, HSV-1 strain F, HSV-1 strain KOS, or HSV-1 strain JS1. In some embodiments, a recombinant oncolytic HSV that includes a genetic construct for expressing an aROR1/aCD3 BspAb as provided herein does not include a functional ICP34.5-encoding gene, and in some examples, all or a portion of the ICP34.5-encoding gene may be deleted. For example, the recombinant oncolytic HSV may be derived from the HSV
17 strain, and the nucleic acid construct encoding an aROR1/aCD3 BspAb may be inserted into the ICP34.5-encoding gene locus.
[0016] In certain embodiments a recombinant oncolytic virus comprising a nucleic acid construct comprising a nucleic acid sequence encoding an aROR1/aCD3 BspAb can further include a nucleic acid sequence encoding a cytokine. For example, an oncolytic virus can include a gene encoding an aROR1/aCD3 BspAb and a gene encoding IL-12. In particular examples disclosed herein an oncolytic virus includes a gene encoding an aROR1/aCD3 BspAb, such as any disclosed herein and a gene encoding IL-12, such as, for example, a human IL-12 having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to SEQ ID NO:46. In some examples, an oncolytic virus can include a gene encoding an aROR1/aCD3 BspAb and a gene encoding a different antibody, for example, an scFv that binds a growth factor or growth factor receptor, such as VEGFR2. In particular examples disclosed herein an oncolytic virus includes a gene encoding an aROR1/aCD3 BspAb, such as any disclosed herein and a gene encoding an anti-VEGFR2 scFv, such as, for example, an VEGFR2 scFV having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:49. In some embodiments, an oncolytic virus as disclosed herein encodes 1) an aROR1/aCD3 BspAb; 2) an IL-12 polypeptide, and 3) an anti-VEGFR2 scFv.
[0017] Further included is a pharmaceutical composition comprising a recombinant oncolytic virus, which may be a recombinant oncolytic HSV, that includes a genetic construct for expressing an aROR1/aCD3 BspAb, and, optionally, one or more additional transgenes, and a pharmaceutically acceptable excipient. The oncolytic virus can be provided in a saline solution, for example, such as PBS, Ringer's, or HBSS, and the formulation can optionally further include, as nonlimiting examples, one or more preservatives, or cryoprotectants (e.g., DMSO or glycerol). In some embodiments, the concentration of virus in the pharmaceutical composition is at least 106 per ml, at least 107 per ml, at least 5 x 107 per ml, or at least 108 per ml.
[0018] Yet another aspect is a method of treating cancer by administering an oncolytic virus encoding an aROR1/aCD3 BspAb as provided herein, including a pharmaceutical composition as provided herein. The oncolytic virus can 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. The subject can be a subject diagnosed with cancer which may be a hematological cancer or a solid tumor. The subject can be, as nonlimiting examples, a dog, horse, or primate, and may be a human subject.
The oncolytic virus can be an oncolytic HSV, and administration may be for example, intravenous, intra-arterial, intracavitary, intraperitoneal, intratumoral, or peritumoral delivery. For example, the oncolytic virus can be delivered by injection, by infusion, or by means of a catheter. The methods can include multiple administrations, where dosings can be separated by days, weeks, or months.
[0019] Also provided are methods of treating a subject using a VFCM produced by culturing cells infected with any of the oncolytic viruses disclosed herein.
[0020] Also provided herein are host cells infected with an oncolytic virus that includes a genetic construct for expressing an aROR1/aCD3 BspAb as provided herein. The host cells can be, for example, mammalian host cells and can be of a cell line. In some embodiments the host cells are Vero cells, BHK cells, or HEK293 cells. Also provided are methods of treating a subject having cancer using a VFCM produced by culturing cells infected with any of the oncolytic viruses disclosed herein. The VCFM can be prepared by for example, centrifugation and filtration of the cell supernatant, where the VFCM can comprise one or more recombinant polypeptides encoded by the oncolytic virus, such as, for example, an aROR1/aCD3 BspAb as provided herein, and optionally IL-12 and/or an anti-antibody. The subject to be treated in some embodiments can be a nonhuman subject.
[0021] Provided in a further aspect are methods for producing a bispecific antibody, including producing any of the aROR1/aCD3 bispecific antibodies disclosed herein, by culturing a host cell infected with an oncolytic virus that includes a genetic construct for expressing a bispecific antibody to produce a virus free conditioned cell medium (VFCM) that includes bispecific antibodies and isolating bispecific antibodies from the VFCM. The VFCM can include one or more additional polypeptides or antibodies, such as but not limited to an IL-12 polypeptide and/or an antibody that binds VEGFR2. Further included are pharmaceutical compositions including aROR1/aCD3 bispecific antibodies as disclosed herein and methods of treating a subject with cancer by administering an aROR1/aCD3 bispecific antibody as disclosed herein to the subject. In some embodiments the methods include treating a subject, such as but not limited to a non-human subject, with a VFCM that may be prepared from cell culture using for example, centrifugation and filtration.
DESCRIPTION OF THE FIGURES
[0022] Figure 1 is a schematic showing an example of a construct encoding an anti-ROR1/anti-CD3 bispecific antibody (transcribed from right to left).
[0023] Figure 2A illustrates the format of an EL1SA detection assay for anti-ROR1/anti-CD3 bispecific antibodies.

[0024] Figure 2B provides binding curves of anti-ROR1/anti-CD3 bispecific antibodies produced by cells infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
VFCMs of cell cultures were assayed.
[0025] Figure 3A illustrates the format of a cell binding assay for anti-ROR1/anti-CD3 bispecific antibodies. Wild type A549 cells express ROR1; A549 cells knocked out for the ROR1 gene were also tested as controls.
[0026] Figure 3B provides the results cell binding assay for anti-ROR1/anti-bispecific antibodies. VFCMs of cultures of cells infected with anti-ROR1/anti-CD3 BspAb-encoding viruses SepGI-189, SepGI-201, and SepGI-203 were assayed.
[0027] Figure 4A illustrates the format of a T cell-tumor cell interaction assay.
[0028] Figure 4B provides the results of flow cytometry analysis of T cell-tumor cell interaction as mediated by aROR1/aCD3 BsAbs present in VFCMS of cultures infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
[0029] Figure 5A illustrates the format of a luciferase-based cell signaling assay for anti-ROR1/anti-CD3 bispecific antibodies.
[0030] Figure 5B provides the results of the cell signaling assay using VFCMS
of cultures infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
[0031] Figure 6 provides percent killing in cytotoxicity assays that included T cells and VFCMS of cultures infected with HSVs SepGI-189, SepGI-201, and SepGI-203.
Interferon gamma (IFNy) secretion by the T cells is also provided in the graphs on the right.
[0032] Figure 7A provides a graph of binding of anti-ROR1 antibody to A549, A549/ROR1 KO, MCF-7, and HepG2 tumor cells.
[0033] Figure 7B provides a graph of percent killing in cytotoxicity assays using A549, MCF-7, and HepG2 tumor cells as targets that included T cells and VFCM of cultures infected with the SepGI-201 HSV that expresses an aROR1/aCD3 BsAb. Controls included assays in the absence of T cells and assays of VFCM produced from cells infected with a SepGI-Null virus that did not express an aROR1/aCD3 BsAb. Also provided are the results of IFNy assays of the co-cultures.
[0034] Figure 8A provides the procedure for assays for killing of A549 tumor cells by aROR1/aCD3 BsAb-expressing HSVs.
100351 Figure 8B provides graphs demonstrating enhanced killing of ROR1-positive tumor cells and ROR1-knockout cells by virus used to infect the cultures at various MOIs.
[0036] Figure 9A illustrates the format of an ELISA detection assay for binding of mouse ROR1 by anti-ROR1/anti-CD3 bispecific antibodies.

[0037] Figure 9B provides the binding curves for antibodies slO and j1v1011 against mouse RORI.
[0038] Figure 10A provides the tumor inoculation and treatment schedule for an in vivo study of treatment of tumors with HSVs SepGI-189 and SepGI-201.
[0039] Figure 10B provides graphs of tumor volumes of A549 tumor-inoculated mice treated with HSVs SepGI-189 and SepGI-201.
[0040] Figure 10C provides a graph of the percent tumor growth inhibition of mice treated with HSVs SepGI-Null, SepGI-189, and SepGI-201.
[0041] Figure 10D provide graphs of body weights over the course of the study shown in Figures 10A, B and C.
[0042] Figure 11A is a schematic showing an example of a construct encoding an anti-ROR1/anti-CD3 bispecific antibody (transcribed from right to left) and a human polypeptide (transcribed from left to right).
[0043] Figure 11B is a schematic showing an example of a construct encoding an anti-ROR1/anti-CD3 bispecific antibody (transcribed from right to left), and an anti-VEGFR2 scFv and human 1L-12 polypeptide. The anti-VEGFR2 scFv and human 1L-12 polypeptide are transcribed from left to right by the same promoter and their coding sequences are connected via a T2A self-cleaving peptide-encoding sequence.
[0044] Figure 12A provides the results of ELISAs for detecting the anti-RSV
antibody in VFCMs of cells infected with different HSVs. The graph shows that the SepGI-207 and SepGI-218 VFCMs included the anti-RSV antibody.
[0045] Figure 12B provides the results of ELISAs for detecting the anti-RORI
antibody in VFCMs of cells infected with different HSVs. The graph shows that the SepGI-201, SepGI-212, and SepGI-216 VFCMs included the anti-ROR1 antibody.
[0046] Figure 12C provides the results of ELISAs for detecting human IL-12 in VFCMs of cells infected with different HSVs. The graph shows that the SepGI-212, SepGI-216, and SepGI-218 VFCMs included human IL-12.
[0047] Figure 13 provides the results of ELISAs for detecting the anti-VEGFR2 antibody in VFCMs of cells infected with different HSVs. The graph shows that the VFCM
of an isolate of SepGI-212 included the anti-VEGFR2 antibody.
100481 Figure 14 is a bar graph providing the results of assays to detect the activity of IL-12 in the VFCMs of cells infected with HSVs SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214, SepGI-216, and SepGI-218.
[0049] Figure 15A provides the results of flow cytometry of unlabeled tumor cells.

[0050] Figure 15B provides the results of flow cytometry of tumor cells labeled with eFluor 450.
[0051] Figure 15C provides the results of flow cytometry of human T cells labeled with eFluor 670.
[0052] Figure 15D provides the results of flow cytometry of labeled tumor cells and labeled T cells co-incubated with VFCM that included an anti-ROR1-anti-CD3 bispecific antibody.
[0053] Figure 16A provides a bar graph of the results of flow cytometry assays for tumor cell-T cell interaction mediated by SepGI-218 VFCM (aRSV-aCD3 bsp antibody plus IL-12) as percentages of analyzed cells when the tumor cells were Hepa 1-6, A549, and knockout cells.
[0054] Figure 16B provides a bar graph of the results of flow cytometry assays for tumor cell-T cell interaction mediated by SepGI-201 VFCM (aROR1-aCD3 bsp antibody) as percentages of analyzed cells when the tumor cells were Hepa 1-6 and A549 cells.
[0055] Figure 16C provides a bar graph of the results of flow cytometry assays for tumor cell-T cell interaction mediated by SepG1-216 VFCM (aROR1-aCD3 bsp antibody plus IL-12 and VEGFR2 antibody) as percentages of analyzed cells when the tumor cells were Hepa 1-6 and A549 cells.
[0056] Figure 17A is a graph showing the percentages of live CD3+ T cells used in T cell activation assays over 3 days, where the T cells have been incubated in the presence of ROR1 knockout tumor target cells (for bars proceeding 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 of each pair of bars provides the value for the assays performed at a 10:1 E:T ratio, and the second of each pair of bars provides the value for the assays performed at a 5:1 E:T
ratio.
100571 Figure 17B is a graph providing the CD3+CD4+ cell count in each of the T cell activation assays. Assay VFCMs and E:T ratios are as in Figure 17A.
[0058] Figure 17C provides the CD25+ T cells as percentages of CD3+CD4+ cells in the activation assays in which the T cells have been incubated in the presence of ROR1+ tumor target cells (for bars proceeding from left to right for each day): VFCM of uninfected cells, VFCM of cells infected with SepGI-Null, VFCM of cells infected with SepG1-207, VFCM of cells infected with SepGI-201, and CD3/CD28 beads. The first of each pair of bars provides the value for the assays performed at a 10:1 E:T ratio, and the second of each pair of bars provides the value for the assays performed at a 5:1 E:T ratio.

[0059] Figure 17D provides the CD69+ T cells as percentages of CD3+CD4+ cells in the activation assays in which the T cells have been incubated in the presence of ROR1+ tumor target cells (for bars proceeding 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 of each pair of bars provides the value for the assays performed at a 10:1 E:T ratio, and the second of each pair of bars provides the value for the assays performed at a 5:1 E:T ratio [0060] Figure 17E is a graph showing the percentages of live CD3+ T cells used in T cell activation assays over 3 days, where the T cells have been incubated in the presence of ROR1+ tumor target cells (for bars proceeding 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 of each pair of bars provides the value for the assays performed at a 10:1 E:T ratio, and the second of each pair of bars provides the value for the assays performed at a 5:1 E:T
ratio.
[0061] Figure 17F is a graph providing the CD3+CD4+ cell count in each of the T cell activation assays. Assay VFCMs and E:T ratios are as in Figure 17E.
[0062] Figure 17G provides the CD25+ T cells as percentages of CD3+CD4+ cells in the activation assays in which the T cells have been incubated in the presence of ROR1+ tumor target cells (for bars proceeding 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 of each pair of bars provides the value for the assays performed at a 10:1 E:T ratio, and the second of each pair of bars provides the value for the assays performed at a 5:1 E:T ratio.
[0063] Figure 17H provides the CD69+ T cells as percentages of CD3+CD4+ cells in the activation assays in which the T cells have been incubated in the presence of ROR1+ tumor target cells (for bars proceeding 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 of each pair of bars provides the value for the assays performed at a 10:1 E:T ratio, and the second of each pair of bars provides the value for the assays performed at a 5:1 E:T ratio 100641 Figure 18A are graphs showing the percentages of live CD3+ T cells used in T cell activation assays over 3 days, where the T cells have been incubated in the presence of A549 wild type (ROR1+) tumor target cells (for bars proceeding from left to right for each day):
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-216. Assays performed at a5:1 E:T ratio.
[0065] Figure 18B are graphs showing the percentages of live CD3+ T cells used in T cell activation assays over 3 days, where the T cells have been incubated in the presence of A549 ROR1 knockout tumor target cells (for bars proceeding from left to right for each day):
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-216. Assays performed at a 5:1 E:T ratio.
[0066] Figure 19A is a graph showing the results of luciferase-based toxicity assays in the absence and presence of T cells where the targets were A549 wild-type cells expressing luciferase and the assays were performed in the presence of VFCMs of uninfected cells or cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214, SepGI-216, and SepGI-218.
[0067] Figure 19B is a graph showing the results of luciferase-based toxicity assays in the absence and presence of T cells where the targets were A549 ROR1 knockout cells expressing luciferase and the assays were performed in the presence of VFCMs of uninfected cells or cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214, SepGI-216, and SepGI-218.
[0068] Figure 19C is a graph providing the percentage killing of the assays of Figure 17C.
[0069] Figure 19D is a graph providing the percentage killing of the assays of Figure 17B.
[0070] Figure 20 shows the cell index over time of cells in impedance-based cytotoxicity assays using A549 wild type cells as targets. See Example 18.
[0071] Figure 21 shows the cell index over time of cells in impedance-based cytotoxicity assays using A549 knockout cells as targets. See Example 18.
DETAILED DESCRIPTION
[0072] Throughout this application various publications, patents, and/or patent applications are referenced. The disclosures of the publications, patents and/or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.
Definitions:
[0073] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise.
Generally, terminologies pertaining to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. 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 indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). A
number of basic texts describe standard antibody production processes, including, Borrebaeck (ed) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995;
McCafferty et al. Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England, 1996; and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, N.J., 1995;
Paul (ed.), Fundamental Immunology, Raven Press, N.Y, 1993; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989)Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY; Stites 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, N.Y., 1986, and Kohler and Milstein Nature 256: 495-497, 1975. All of the references cited herein are incorporated herein by reference in their entireties. Enzymatic reactions and enrichment/purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art.
Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
[0074] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.
[0075] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms -a-, "an- and "the-, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.
[0076] It is understood the use of the alternative (e.g., "or") herein is taken to mean either one or both or any combination thereof of the alternatives.

[0077] The term "and/or" used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B,"
"A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C- is intended to encompass each of the following aspects: 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).
[0078] As used herein, terms -comprising", -including", -having" and -containing", and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language -comprising," otherwise analogous aspects described in terms of -consisting of' and/or "consisting essentially of" are also provided.
[0079] As used herein, the term -about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about- or "approximately- can mean within one or more than one standard deviation per the practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10%
(i.e., 10%) or more depending on the limitations of the measurement system.
For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of "about" or "approximately"
should be assumed to be within an acceptable error range for that particular value or composition.
[0080] The terms "peptide", "polypeptide" and "protein" and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids.
Polypeptides include recombinant or chemically-synthesized forms. Polypeptides also include precursor molecules that have not yet been subjected to cleavage, for example cleavage by a secretory signal peptide or by non-enzymatic cleavage at certain amino acid residues. Polypeptides include mature molecules that have undergone cleavage.
These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as

13 muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins. Two or more polypeptides (e.g., 3 polypeptide chains) can associate with each other, via covalent and/or non-covalent association, to form a polypeptide complex. Association of the polypeptide chains can also include peptide folding. Thus, a polypeptide complex can be dimeric, trimeric, tetrameric, or higher order complexes depending on the number of polypeptide chains that form the complex.
[0081] The terms -nucleic acid", "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically-synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA
molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof Nucleic acid molecule can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment or scFv, derivative, mutein, or variant thereof In one embodiment, nucleic acids comprise one type of polynucleotide or a mixture of two or more different types of polynucleotides. Nucleic acids encoding bispecific antibodies are described herein.
[0082] The term "recover" or -recovery" or "recovering", and other related terms, refers to obtaining a protein (e.g., an antibody or an antigen binding portion thereof), from host cell culture medium or from host cell lysate or from the host cell membrane. In one embodiment, the protein is expressed by the host cell as a recombinant protein fused to a secretion signal peptide (leader peptide sequence) sequence which mediates secretion of the expressed protein from a host cell (e.g., from a mammalian host cell). The secreted protein can be recovered from the host cell medium. In one embodiment, the protein is expressed by the host cell as a recombinant protein that lacks a secretion signal peptide sequence which can be recovered from the host cell lysate. In one embodiment, the protein is expressed by the host cell as a membrane-bound protein which can be recovered using a detergent to release the expressed protein from the host cell membrane. In one embodiment, irrespective of the method used to recover the protein, the protein can be subjected to procedures that 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 or any combination or two or more procedures including

14 affinity chromatography, hydroxyapatite chromatography, ion-exchange chromatography, reverse phase chromatography and/or chromatography on silica. In one embodiment, affinity chromatography comprises protein A or G (cell wall components from Staphylococcus aureus).
[0083] The term "isolated" refers to a protein (e.g., an antibody or an antigen binding portion thereof) or polynucleotide that is substantially free of other cellular material. A
protein may be rendered substantially free of naturally associated components (or components associated with a cellular expression system or chemical synthesis methods used to produce the antibody) by isolation, using protein purification techniques well known in the art. The term isolated also refers to protein or polynucleotides that are substantially free of other molecules of the same species, for example other protein or polynucleotides having different amino acid or nucleotide sequences, respectively. The purity of homogeneity of the desired molecule can be assayed 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 present disclosure are isolated.
[0084] The term "signal peptide", "[peptide] signal sequence", "leader sequence", -leader peptide-, or "secretion signal peptide- refers to a peptide sequence that is located at the N-terminus of a polypeptide. A leader sequence directs a polypeptide chain to a cellular secretory pathway and can direct integration and anchoring of a membrane protein into the lipid bilayer of the cellular membrane. Typically, a leader sequence is about 10-60 amino acids in length, more commonly 15-50 amino acids in length. A leader sequence can direct transport of a precursor polypeptide from the cytosol to the endoplasmic reticulum. In various embodiments, a leader sequence includes signal sequences comprising CD8a, CD28, or CD16 leader sequences or a mouse or human Ig gamma secretion signal peptide.
In one embodiment, a leader sequence comprises a mouse Ig gamma leader peptide sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO:).
[0085] An "antigen binding protein" and related terms used herein refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol.
Prog. 20:639-654. In addition, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics utilizing fibronection components as a scaffold.
[0086] An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin. In one embodiment, an "immunoglobulin" refers to a naturally-occurring tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 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 function.
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 1gM, 1gD, IgG, IgA, and IgE, respectively. Within 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 "D"
region of about 10 more amino acids. See generally, Fundamental Immunology Ch.
7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The heavy and/or light chains may or may not include a leader sequence for secretion. The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites. In one embodiment, an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens. For example, a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules. In various embodiments, bispecific antibodies of the present disclosure exhibit immunoglobulin-like properties and bind specifically to two different target antigens (ROR1 and CD3).
100871 The variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarily determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0088] One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.
[0089] The assignment of amino acids to each domain is in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US
Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991 (-Kabat numbering"). Other numbering systems for the amino acids in immunoglobulin chains include IMGT® (international ImMunoGeneTics information system; Lefranc et al, Dev.
Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, I 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.
[0090] An "antibody" and "antibodies" and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof that binds specifically to an antigen.
Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alio, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarily determining region (CDR) fragments. single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
[0091] Antibodies include recombinantly produced antibodies and antigen binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies.
Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, 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 fragment (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies. Antibodies include monoclonal and polyclonal populations. In some embodiments described herein, bispecific antibodies include two single chain variable fragment antibodies, which may be described as "scFv moieties" or simply -scFvs" of the bispecific antibody molecule, joined by a linker.

[0092] An "antigen binding domain," "antigen binding region," or "antigen binding site"
and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR
domains. Antigen binding domains from monoclonal antibodies and bispecific antibodies are provided herein.
[0093] The terms "specific binding", "specifically binds" or "specifically binding" and other related terms, as used herein in the context of an antibody or antigen binding protein (e.g., heterodimeric antibody) or antibody fragment, refer to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens). In one embodiment, an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10-5 M or less, or 10' M or less, or 10-7 M or less, or 10' M or less, or 10 M or less, or 10-ll) M or less, or 10-'' M or less. Bispecific antibodies that specifically bind ROR1 and CD3 are described herein.
[0094] In one embodiment, binding specificity can be measure by ELISA, radioimmune assay (MA), electrochemiluminescence assays (ECL), immunoradiometric assay (IRMA), or enzyme immune assay (ETA).
[0095] In one embodiment, a dissociation constant (KD) can be measured using a BIACORE surface plasmon resonance (SPR) assay. Surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
[0096] An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof). An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein. An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen's primary sequence but that, in the context of the antigen's tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
Generally, the variable regions, particularly the CDRs, of an antibody interact with the epitope.
Bispecific antibodies that bind an epitope of a ROR1 polypeptide and that bind an epitope of a CD3 polypeptide are described herein.

[0097] An "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody", or "antigen-binding portion of an antibody" and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(a131)2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g.
scFv);
polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide. Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
Antigen binding portions include, inter alia, Fab, Fab', F(a13)2, Fv, domain antibodies (dAbs), and complementarily determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
[0098] The terms -Fab", -Fab fragment" and other related terms refers to a monovalent fragment comprising a variable light chain region (VL), constant light chain region (CL), variable heavy chain region (VII), and first constant region (Cm). A Fab is capable of binding an antigen. An F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. A F(Ab')2 has antigen binding capability.
An Fd fragment comprises Vti and CHI regions. An Fv fragment comprises VL and Vit regions. An FAT can bind an antigen. A dAb fragment has a Vfi domain, a VL
domain, or an antigen-binding fragment of a VH or VL domain (U.S. Patents 6,846,634 and 6,696,245; U.S.
published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et al., Nature 341:544-546, 1989).
[0099] A single-chain antibody (scFv) is an antibody in which a VL and a VII region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain. Preferably the linker is long enough to allow the protein chain to fold back on 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).
Single chain antibodies that specifically bind ROR1 and single chain antibodies that specifically bind CD3 are described herein.
1001001 Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and V. domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., 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 a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
[00101] The term "human antibody- refers to antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). These antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes.
1001021 A -humanized" antibody refers to an antibody having a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of 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 changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen.
Examples of how to make humanized antibodies may be found in U.S. Pat. Nos.
6,054,297, 5,886,152 and 5,877,293.
1001031 The term -chimeric antibody" and related terms 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 of the CDRs are derived from a human antibody. In another embodiment, all of the CDRs are derived from a human antibody. In another embodiment, the CDRs from more than one human antibody are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody.
In another example, the CDRs originate from different species such as human and mouse, or human and rabbit, or human and goat. One skilled in the art will appreciate that other combinations are possible.
[00104] Further, the framework regions may be derived from one of the same antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody (-ies) from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind a target antigen).
[00105] As used herein, the term "variant" polypeptides and "variants" of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.
[00106] As used herein, the term "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. 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.
1001071 The term -Fc" or -Fc region" as used herein refers to the portion of an antibody heavy chain constant region beginning in or after the hinge region and ending 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 a half Fc region can dimerize to form an Fc region. An Fc region can bind Fe cell surface receptors and some proteins of the immune complement system. An Fc region exhibits effector function, including any one or any combination of two or more activities including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and/or cell binding. An Fc region can bind an Fc receptor, including FcyRI (e.g., CD64), FcyRII
CD32) and/or FcyRIII (e.g., CD16a).
[00108] The term -labeled antibody" or related terms as used herein refers to antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A. A variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens). Any of the bispecific antibodies described herein can be unlabeled or can be joined to a detectable label or moiety.
[00109] The -percent identity" or "percent homology" and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences. A comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology"
of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. Expressions such as "comprises a sequence with at least X% identity to Y" with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y.

[00110] In one embodiment, the amino acid sequence of a test antibody may be similar but not identical to any of the amino acid sequences of the polypeptides that make up the bispecific antibodies described herein. The similarities between the test antibody and the polypeptides can be at least 95%, or at or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, to any of the polypeptides that make up the bispecific antibodies described herein. In one embodiment, similar polypeptides can contain amino acid substitutions within a heavy and/or light chain. In one embodiment, the amino acid substitutions comprise one or more conservative amino acid substitutions.
A
"conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference in its entirety. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl 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:
aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
[00111] Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins having varied antigenic specificity. If such antibodies are subjected to affinity purification, they can be enriched for a particular antigenic specificity. Such enriched preparations of antibodies usually are made of less than about 10% antibody having specific binding activity for the particular antigen. Subjecting these preparations to several rounds of affinity purification can increase the proportion of antibody having specific binding activity for the antigen. Antibodies prepared in this manner are often referred to as "monospecific."
Monospecific antibody preparations can be made up of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specific binding activity for the particular antigen. Antibodies can be produced using recombinant nucleic acid technology as described below.

[00112] A "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene). Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell). Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector. Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct. Vectors can be single-stranded or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. A
donor nucleic acid used for gene editing methods employing zinc finger nuclease, TALEN or CRISPR/Cas can be a type of a vector. One type of vector is a "plasmid," which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene. A viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome. Examples of viral vectors include retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors.
Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
[00113] An "expression vector is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers.
Expression vectors can include ribosomal binding sites and/or polyadenylation sites.
Expression vectors can include one or more origin of replication sequence. Regulatory sequences direct transcription, or transcription and translation, of a transgene linked to the expression vector which is transduced into a host cell. The regulatory sequence(s) can control the level, timing and/or location of expression of the transgene. The regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid).
Regulatory sequences can be part of a vector. Further examples of regulatory sequences are described in, for example, 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 can comprise nucleic acids that encode at least a portion of any of the bispecific antibodies described herein.
[00114] A transgene is -operably linked" to a vector when there is linkage between the transgene and the vector to permit functioning 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 (e.g., the level, timing, or location of expression) of the transgene.
[00115] The terms "transfected" or "transformed" or "transduced" or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene). The host cell includes the primary subject cell and its progeny.
Exogenous nucleic acids encoding at least a portion of any of the bispecific antibodies described herein can be introduced into a host cell. Expression vectors comprising at least a portion of any of the bispecific antibodies described herein can he introduced into a host cell, and the host cell can express polypeptides comprising at least a portion of the bispecific antibodies.
[00116] The terms "host cell" or "or a population of host cells" or related terms as used herein refer to a cell (or a population thereof) into which foreign (exogenous or transgene) nucleic acids have been introduced. The foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and/or polypeptide encoded by the foreign nucleic acid (transgene). A host cell (or a population thereof) can be a cultured cell or can be extracted from a subject.
The host cell (or a population thereof) includes the primary subject cell and its progeny without any regard for the number of passages. Progeny cells may or may not harbor identical genetic material compared to the parent cell. Host cells encompass progeny cells. In one embodiment, a host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and/or manipulated in any way to express an antibody, as disclosed herein. In one example, the host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or an antigen binding portion thereof, described herein. Host cells and populations thereof can harbor an expression vector that is stably integrated into the host's genome or can harbor an extrachromosomal expression vector. In one embodiment, host cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.

[00117] Transgenic host cells can be prepared using non-viral methods, including well-known designer nucleases including zinc finger nucleases, TALENS or CRISPR/Cas. A
transgene can be introduced into a host cell's genome using genome editing technologies such as zinc finger nuclease. A zinc finger nuclease includes a pair of chimeric proteins each containing a non-specific endonuclease domain of a restriction endonuclease (e.g., FokI ) fused to a DNA-binding domain from an engineered zinc finger motif The DNA-binding domain can be engineered to bind a specific sequence in the host- s genome and the endonuclease domain makes a double-stranded cut. The donor DNA carries the transgene, for example any of the nucleic acids encoding a CAR or DAR construct described herein, and flanking sequences that are homologous to the regions on either side of the intended insertion site in the host cell's genome. The host cell's DNA repair machinery enables precise insertion of the transgene by homologous DNA repair. Transgenic mammalian host cells have been prepared using zinc finger nucleases (U.S. patent Nos. 9,597,357, 9,616,090, 9A416,074 and 8.)45,863). A transgenic host cell can be prepared using TALEN
(Transcription Activator-Like Effector Nucleases) which are similar to zinc finger nucleases in that they include a non-specific endonuclease domain fused to a DNA-binding domain which can deliver precise transgene insertion. Like zinc finger nucleases, TALEN also introduce a double-stranded cut into the host's DNA. Transgenic host cells can be prepared using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).
CRISPR
employs a Cas endonuclease coupled to a guide RNA for target specific donor DNA
integration. The guide RNA includes a conserved multi-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region in the target DNA and hybridizes to the host cell target site where the Cas endonuclease cleaves the double-stranded target DNA. The guide RNA can be designed to hybridize to a specific target site. Similar to zinc finger nuclease and TALEN, the CRISPR/Cas system can be used to introduce site specific insertion of donor DNA having flanking sequences that have homology to the insertion site. Examples of CRISPRJCas systems used to modify genomes are described for example in U.S. Pat. Nos. 8,697,359, 10,000,772, 9,790,490, and U. S. Patent Application Publication No. US 2018/0346927. In one embodiment, transgenic host cells can be prepared using zinc finger nuclease, TALEN or CRISPR/Cas system, and the host target site can be a TRAC gene (T Cell Receptor Alpha Constant). The donor DNA can include for example any of the nucleic acids encoding a CAR or DAR construct described herein.
Electroporation, nucleofection or lipofection can be used to co-deliver into the host cell the donor DNA with the zinc finger nuclease, TALEN or CRISPR/Cas system.

1001181 A host cell can be a prokaryote, for example, E. coil, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. In one embodiment, a host cell can be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody thereby generating a transfected/transformed host cell which is cultured under conditions suitable for expression of the antibody by the transfected/transformed host cell, and optionally recovering the antibody from the transfected/transformed host cells (e.g., recovery from host cell lysate) or recovery from the culture medium. In one embodiment, host cells comprise non-human cells including CHO, BHK, NSO, SP2/0, and YB2/0.
In one embodiment, host cells comprise human cells including HEK293, HT-1080, Huh-7 and PER.C6. Examples of host cells include the COS-7 line 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 which grow in serum- free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL
10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (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 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, host cells include lymphoid cells such as YO, NSO or Sp20. In one embodiment, a host cell is a mammalian host cell, but is not a human host cell. Typically, a host cell is a cultured cell 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" can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell, or a population of host cells, harboring a vector (e.g., an expression vector) operably linked to at least one nucleic acid encoding one or more bispecific antibodies are described herein.
[00119] Polypeptides of the present disclosure (e.g., antibodies and antigen binding proteins) can be produced using any method known in the art. In one example, the polypeptides are produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector which is introduced into a host cell and expressed by the host cell under conditions promoting expression.
[00120] General techniques for recombinant nucleic acid manipulations are described for example in Sambrook et al., in Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., in Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodic updates, herein incorporated by reference in their entireties. The nucleic acid (e.g., DNA) encoding the 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 a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA
ribosomal binding sites, and sequences that control the termination of transcription and translation. The expression vector can include an origin or replication that confers replication capabilities in the host cell. The expression vector can include a gene that confers selection to facilitate recognition of transgenic host cells (e.g., transformants).
[00121] The recombinant DNA can also encode any type of protein tag sequence that may be useful for purifying the protein. Examples of protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).
[00122] The expression vector construct can be introduced into the host cell using a method appropriate for the host cell. A variety of methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; microprojectile bombardment;
lipofection; and infection (e.g., where the vector is an infectious agent).
Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.

[00123] Suitable bacteria include gram negative or gram positive organisms, for example, E. coil or Bacillus spp. Yeast, preferably from the Saccharom.yces species, such as S. cerevisiae, may also be used for production of polypeptides. Various mammalian or insect cell culture systems can also be employed to express recombinant proteins.
Baculovirus systems for production of heterologous proteins in insect cells are reviewed by 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, 2931, and BHK
cell lines. Purified polypeptides are prepared by culturing suitable host/vector systems to express the recombinant proteins. For many applications, the small size of many of the polypeptides disclosed herein would make expression in E. coil as the preferred method for expression. The protein is then purified from culture media or cell extracts.
Any of the bispecific antibodies disclosed herein can be expressed by transgenic host cells.
[00124] Antibodies and antigen binding proteins disclosed herein can also be produced using cell-translation systems. For such purposes the nucleic acids encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial cell-free translation system.
[00125] Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically. Codon usage may be selected so as to improve expression in a cell.
Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et al., Proc. Natl.
Acad. Sci.
USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Puff. 2002 (1):96-105, Connell N
D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev.

60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.
[00126] Antibodies and antigen binding proteins described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the protein can also be produced by chemical synthesis.
[00127] Antibodies and antigen binding proteins described herein can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry.
Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations of these.
After purification, polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.
[00128] The 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 numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any of the bispecific antibodies described herein can be expressed by transgenic host cells and then purified to about 65-98% purity or high level of purity using any art-known method.
[00129] In certain embodiments, the antibodies and antigen binding proteins herein can further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of a polypeptide side chain or of a hydrophobic group. As a result, the modified polypeptides may contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates.
A preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to the polypeptide. Sialic acid moieties improve 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.
[00130] In one embodiment, the antibodies and antigen binding proteins described herein can be modified to become soluble polypeptides which comprises linking the Antibodies and antigen binding proteins to non-proteinaceous polymers. In one embodiment, the non-proteinaceous polymer comprises polyethylene glycol ("PEG--), polypropylene glycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos. 4,640,835;
4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
1001311 The present disclosure provides therapeutic compositions comprising any of the bispecific antibodies described herein in an admixture with a pharmaceutically-acceptable excipient. An excipient encompasses carriers, stabilizers, and excipients.
Examples of pharmaceutically acceptable excipients includes for example inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Additional examples include buffering agents, stabilizing agents, preservatives, non-ionic detergents, anti-oxidants, and isotonifiers.
[00132] Therapeutic compositions and methods for preparing them are well known in the art and are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.).
Therapeutic compositions can be formulated for parenteral administration may, and can for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibody (or antigen binding protein thereof) described herein. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the antibody (or antigen binding protein thereof). Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the antibody (or antigen binding protein thereof) in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.
[00133] Any of the bispecific antibodies (or antigen binding protein thereof) may be optionally administered as a pharmaceutically acceptable salt. such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. In one example, the antibody (or antigen binding protein thereof) is formulated in the presence of sodium acetate to increase thermal stability.
[00134] Any of the bispecific antibodies (or antigen binding protein thereof) may be formulated for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.

[00135] The term "subject" as used herein refers to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
[00136] The term "administering-, "administered- and grammatical variants refers to the physical introduction of an agent to a subject, using any of the various 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, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. Any of the bispecitic antibodies described herein (or antigen binding protein thereof) can be administered to a subject using art-known methods and delivery routes.
[00137] The terms "effective amount", "therapeutically effective amount" or "effective dose" or related terms may be used interchangeably and refer to an amount of antibody or an antigen binding protein (e.g., bispecific antibodies) that when administered to a subject, is sufficient to effect a measurable improvement or prevention of a disease or disorder associated with tumor or cancer antigen expression. Therapeutically effective amounts of antibodies provided herein, when used alone or in combination, will vary depending upon the relative activity of the antibodies and combinations (e.g. , in inhibiting cell growth) and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[00138] In one embodiment, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In general, the polypeptide is administered at about 0.01 g/kg to about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1 mg/kg to about 20 mg/kg per day. The polypeptide may be administered daily (e.g., once, twice, three times, or four times daily) or preferably less frequently (e.g., weekly, every two weeks, every three weeks, monthly, or quarterly). In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
[00139] The present disclosure provides methods for treating a subject having a disease associated with expression of one or more tumor-associated antigens. The disease comprises cancer or tumor cells expressing the tumor-associated antigens, such as for example CD38 and/or CD3 antigen. In one embodiment, the cancer or tumor includes cancer of the prostate, breast, ovary, head and neck, bladder, skin, colorectal, anus, rectum, pancreas, lung (including non-small cell lung and small cell lung cancers), leiomyoma, brain, glioma, glioblastoma, esophagus, liver, kidney, stomach, colon, cervix, uterus, endometrium, vulva, larynx, vagina, bone, nasal cavity, paranasal sinus, nasopharynx, oral cavity, oropharynx, larynx, hypolarynx, salivary glands, ureter, urethra, penis and testis.
[00140] In one embodiment, the cancer comprises hematological cancers, including leukemias, lymphomas, myelomas and B cell lymphomas. Hematologic 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 lymphocytic 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, plasmacytoma, giant cell myeloma, plasma cell myeloma, heavy-chain myeloma, light chain or Bence-Jones myeloma, lyinphornatoid granitioinatosis, post-transplant lyinphoproliferative disorder, an imritunorgulatory disorder, dietimatoid ;arthritis, myasthenia. gra.vis, idiopathic .thrombocytopenia purpura, anti-phospholipi d syndrome. Chagas' disease, Grave's disease, 'Wegener's grartulomatosis, pol.!yr-arter4is nodosa, Sjogren's syndrome, pernphigus vulgaris, scleroderrna, multiple sclerosis, anti-phospholipid syndrome, .ANCA associated vaseulitis. Goodpasture's disease, Kawasaki disease, autoimmurie heinelytie anemia and rapidly progressive glomerulonephritis, heavy-chain disease, primary or immunocyte-associated arayloidosis, and monoclonal garnitiopailly of undetermined significance.
Recombinant Oncolytic Viruses encoding anti-RORlianti-CD3 Bispecific Antibodies [00141] The present disclosure provides, inter alia, oncolytic viruses that express bispecific antibodies that bind ROR1 and CD3, cells infected with such viruses, and methods of treating cancer using the viruses expressing bispecific antibodies. Also provided is virus-free conditioned culture media (VFCM) produced by the infected cells and methods of producing pharmaceutical formulations using VFCMs.
[00142] Oncolytic viruses provide a targeted approach to cancer therapy, as they selectively replicate in and lyse tumor cells. Various types of oncolytic viruses are known in the art, including include parvoviruses, 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 cause cell lysis and/or induce an immune response to the tumor cells they infect. This disclosure provides recombinant oncolytic viruses that include a heterologous gene construct that encodes an anti-ROR1/anti-CD3 bispecific antibody (aROR1/aCD3 BspAb) such as any disclosed herein. The construct can include a promoter active in a mammalian cell operably linked to the aROR1/aCD3 BspAb-encoding sequence and the construct can be inserted into the genome of the oncolytic virus.
[00143] In various embodiments, an oncolytic virus modified for expression of an aROR1/aCD3 BspAb can be a herpes simplex virus (Human alphaherpesvirus; HSV), such as an HSV-1. HSV-2, or a recombinant HSV having sequences of both HSV-1 or HSV-2. For example, a laboratory strain or clinical isolate of an HSV-1 or HSV-2 strain can be used.
Multiple isolated and modified strains of HSV-1 and HSV-2 are known in the art and can be considered for use in the compositions and methods disclosed herein, including, as nonlimiting examples, HSV-1 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, 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 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. Also considered for use in the compositions and methods provided herein are derivates or mutants of these strains or others that may be known in the art or isolated.
[00144] Derivatives of viral strains include, without limitation, viruses that may have one or more endogenous genes that is mutated, including one or more endogenous genes that is partially or entirely deleted, may have a transgene (heterologous gene) inserted into the viral genome (including but not limited to one or more selectable markers, negative selectable markers ("suicide genes-), and/or detectable markers (e.g., a gene encoding a fluorescent protein or a gene encoding an enzyme that produces a detectable product)), and/or may have one or more modifications such as but not limited to restriction sites, recombination sites or "landing pads", exogenous promoters, etc. A derivative may have other modifications such as but not limited to deletion or mutation of non-gene sequences, such as for example 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 that alternatively or in addition to other modifications include one or more transgenes supporting or regulating viral growth or viability, one or more genes affecting host cell functions, or one or more transgenes encoding therapeutic proteins, as nonlimiting examples.
1001451 In some nonlimiting embodiments the HSV is an HSV-1 such as HSV-1 strain 17, HSV-1 strain KOS, or HSV-1 strain F, or a derivative of any of HSV-1 strain 17, HSV-1 strain KOS, or HSV-1 strain F. For example, a strain used for the introduction of an ScFA,r-Fc-TGF[3trap construct can be HSV-1 strain 17 mutant 1716. HSV-1 strain F mutant (Chou & Roizman (1992) Proc. Natl. Acad Sc. 89: 3266-3270), HSV-1 strain F
mutant G207 (Toda etal. (1995) Human Gene Therapy 9:2177-2185), HSV-1 strain F mutant (Todo etal. (2001) Proc Natl Acad Sci USA 98:6396-6401), HSV-1 mutant NV1020 (Geevarghese et at. (2010) Human Gene Therapy 21:1119-28), RE6 (Thompson et at. (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 etal. (2010) The Open Virology Journal 4:103-108), or Talimogenelaherparepvec ("TVec", formerly OncoVexk;
Liu et al. (2003) Gene Therapy 10:292-303), or a further derivative or mutant of any of these.
1001461 Mutation of endogenous viral genes can include mutation or deletion of genes that affect replication or propagation of the virus in non-cancerous cells or the ability of viruses to avoid host defenses. For example, an HSV that includes an aROR1/aCD3 BspAb can be deleted in any of the ICP34.5-encoding gene, the ICP6-encoding gene, the ICP0-encoding gene, the vhs-encoding gene, or the ICP27-encoding gene. Mutants that do not produce a functional protein encoded by a gene or genes (where the gene is multicopy) are refen-ed to herein as having a functionally deleted gene. Functional deletion of one or more of the ICP34.5-encoding gene, the ICP6-encoding gene, the 'CPO-encoding gene, and the vhs-encoding gene can result in an HSV impaired in replication in noncancerous cells.
[00147] The ICP34.5-encoding gene RL1 is located in the long repeat (RL) of the HSV-1 genome and is present in two copies. In some embodiments one or both copies of the ICP34.5-encoding genes is mutated or is partially or entirely deleted such that no functional protein is made. In preferred embodiments, an oncolytic HSV that includes a transgene encoding an ScFv-Fc-TGFf3trap protein and, optionally, an IL12 gene, is functionally deleted for the ICP34.5-encoding gene responsible for neurovirulence (Chou etal.
(1990) Science 250:1262-1266), e.g., both copies of the ICP34.5-encoding gene of the HSV
viral genome are inactivated. For example the oncolytic HSV used for introduction of an ScFv-Fc-TGFI3trap construct can be a mutant of HSV-1 strain 17 and may be HSV1716 (Brown etal.
(1994) Journal of General Virology 75: 2367-2377; MacLean et al. (1991) Journal of General Virology 72:631-639) or a mutant or derivative thereof, or may be SeprehvecTM
or a derivative or mutant thereof HSV1716 and SeprehvecTm both have deletions in both copies of the ICP34.5-encoding gene such that they do not produce a functional gene product, but each otherwise has a genome substantially similar to that of HSV strain 17, which has been completely sequenced (Pfaff et al. (2016) J Gen Virol 97:2732-2741;
ncbi.nlm.nih.gov/genome, Accession number JN555585).
[00148] Recombinant HSVs as provided herein can have one or more transgenes inserted into the ICP34.5 locus, the ICP6 locus, the 'CPO locus, or the vhs locus. In some preferred embodiments a recombinant oncolytic HSV as provided herein can have an ctROR1/aCD3 BspAb gene inserted into a deleted ICP34.5-encoding gene locus. In some preferred embodiments a recombinant oncolytic HSV as provided herein is functionally deleted for ICP34.5 (i.e., is ICP34.5 null) and has an aROR1/aCD3 BspAb gene inserted into both copies of the ICP34.5-encoding gene locus.
[00149] The recombinant oncolytic viruses provided herein, which are able to infect many tumor cell types, include expression constructs that encode novel bispecific antibodies that bind ROR1, a protein expressed on many tumor cells, and CD3, expressed on T cells, where the bispecific antibodies can be expressed and secreted by cells infected by the recombinant viruses that encode them. The RORI scFv moiety of the aROR1/aCD3 BspAb specifically binds an immune checkpoint protein and the CD3 scFv moiety binds T cells, bringing T cells into proximity with target tumor cells to enhance killing of tumor cells.

[00150] Exemplary constructs encoding the ctROR1/aCD3 BspAbs described herein use scFvs derived from ROR1 monoclonal antibody oil, having a variable heavy chain region of SEQ ID NO:1 or sequences having at least 95% identity thereto, with heavy chain variable region CDRs of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a variable light chain region of SEQ ID NO:5 or sequences having at least 95% identity thereto, with light chain variable region CDRs of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
[00151] Additional exemplary constructs encoding the aROR1/aCD3 BspAbs described herein use scFvs derived from ROR1 monoclonal antibody s10, having a variable heavy chain region of SEQ ID NO:10 or sequences having at least 95% identity thereto, with heavy chain variable region CDRs of SEQ ID NO: ii, SEQ ID NO:12, and SEQ ID NO:13, and a variable light chain region of SEQ ID NO:14 or sequences having at least 95%
identity thereto, with light chain variable region CDRs of SEQ ID NO:15, SEQ ID NO:16, and SEQ
ID NO:17.
[00152] Further exemplary constructs encoding the aROR1/aCD3 BspAbs described herein use scFvs derived from ROR1 monoclonal antibody jlvl 011, having a variable heavy chain region of SEQ ID NO:19 or sequences having at least 95% identity thereto, with heavy chain variable region CDRs of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, and a variable light chain region of SEQ ID NO:23 or sequences having at least 95%
identity thereto, with light chain variable region CDRs of SEQ ID NO:24, SEQ ID NO:25, and SEQ
ID NO:26.
[00153] In particular examples an ctROR1/aCD3 BspAbs can have the sequence of SEQ
ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or can have an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any of SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40.
[00154] The aROR1/aCD3 BspAbs can have the format: 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 aROR1/aCD3 BspAbs can be N-terminal to the anti-CD3 scFv moiety or vice versa.
[00155] A construct encoding an aROR1/aCD3 BspAb, an IL-12 polypeptide, or an anti-VEGFR antibody (e.g., an anti-VEGFR scFv) can be operably linked to a promoter for expression in a eukaryotic cell. Examples of promoters that can be used in a recombinant virus for expression of an ctROR1/aCD3 BspAb include, without limitation, a Cytomegalovirus (CMV) promoter (e.g., SEQ ID NO:33), a hybrid CMV promoter (e.g., U.S
9,777,290), an HTLV promoter, an EFla promoter, a hybrid EF1ct/HTLV promoter (e.g., SEQ ID NO:32), a JeT promoter (US Patent No. 6,555,674), a SPARC promoter (e.g., US
8,436,160), an RSV promoter, an SV40 promoter, or a retroviral LTR promoter such as an MMLV promoter, or a promoter derived from any of these. The construct can also include a polyadenylation sequence, such as, for example, a BGH, 5V40, HGH, or RBG
polyadenylation sequence. In some embodiments the polyadenylation sequence has the sequence of SEQ ID NO:38.
[00156] Oncolytic viruses, such as for example those described herein, that include trangenes encoding an aROR1/aCD3 BspAb, IL-12, and/or an anti-VEGFR antibody, can be used to infect host cells that can be cultured for the production of VFCMs, and optionally bispecific antibodies or other recombinant polypeptides that may 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. A subject, such as a subject having cancer, can be treated with a VFCM that includes, for example, an aROR1/aCD3 BspAb. The subject in some embodiments can be a non-human animal, and may be, as nonlimiting examples, a dog, horse, cat, monkey, ape, farm animal, or member of an endangered species.
[00157] The disclosure provides methods of treating cancer using a recombinant HSV that encodes an aROR1/aCD3 BspAb. The method can include administering a recombinant HSV
that comprises a nucleic acid construct encoding an aROR1/aCD3 BspAb as provided herein to a subject having cancer. In some embodiments the cancer may be a solid tumor. The recombinant HSV can be any disclosed herein, such as, for example, any that encodes an aROR1/aCD3 BspAb. The subject may be a human or may be a non-human animal such as, for example, a dog, cat, cow, bull, or horse. The cancer can be without limitation, bladder, bone, breast, eye, stomach, head and neck, kidney, liver, lung, ovarian, pancreatic, prostate, skin, or uterine cancer, a mesothelioma, a glioma, a neurocytoma, or a chondrosarcoma. The administering can be by any means and can be, as nonlimiting examples, parenteral, systemic, intracavitary (e.gõ intrapleural, intraperitoneal), peritumoral, or intratumoral, and may be by injection, intravenous or intra-arterial infusion, or other delivery means. Injection can be, for example, parenteral, subcutaneous, intramuscular, intravenous, intra-arterial, intratumoral, or peritumoral. The treatment regimen may include more than one administration of the virus and can include multiple dosings over a period of days, weeks, or months.
[00158] In some embodiments the aROR1/aCD3 BspAb encoded by the HSV used in the methods is an aROR1/aCD3 BspAb having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 (or otherwise homologous aROR1/aCD3 BspAbs having different signal peptides or lacking signal peptides). The HSV can further include one or more additional transgenes that may encode, as nonlimiting examples, an IL-12 polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:47 or an 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 otherwise homologous polypeptideshaving different signal peptides or lacking signal peptides).
EXAMPLES
Example 1. ot-ROR1/a-CD3 BspAb Herpes Simplex Virus (HSV) constructs.
[00159] Three constructs were designed for expressing a bispecific antibody (BspAb) that included, proceeding from the N-terminus to the C-terminus: a signal peptide (SEQ ID
NO:28), an scFv antibody that specifically binds ROR1, a GS linker (SEQ ID
NO:29), and an anti-CD3 scFv antibody (hum291, SEQ ID NO:34). Figure 1 provides a general diagram representing the constructs encoding aR0R1/aCD3 bispecific antibodies (aR0R1/aCD3 BspAbs). All constructs included the EFla/HTLV hybrid promoter (SEQ ID NO:41) operably linked to the BspAb-encoding sequence. Constructs encoding three different aR0R1/aCD3BspAbs were made that differed only in the ROR1 scFv: an aCD3/aR0R1 BspAb that included an oil ROR1 scFv (SEQ ID NO:9), an aCD3/aR0R1 BspAb that included an sl 0 ROR1 scFv (SEQ ID NO:18), and an aCD3/ aR0R1 BspAb that included a j1v1011 ROR1 scFv (SEQ ID N0:27).
[00160] To clone these constructs into a 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 that encodes the y34.5 kd (ICP34.5) polypeptide responsible for neurovirulence are disrupted by a 695 bp deletion (nucleotides 125975 to 125221 within the RL1 sequence) that inactivates the RL1 gene. The RL1 deletion site includes attR recombination sites for insertion of any gene or construct of interest flanked by attL sequences. The anti-CD3/anti-ROR1 BspAb constructs flanked by attL
sequences were inserted into both RL1 loci at the deletion sites using in vitro recombinational cloning that used the LR ClonaseTM Plus enzyme mixture of Integrase and Integration Host Factor (ThermoFisher, Carlsbad, CA) essentially according to the manufacturer's instructions.

[00161] Following the recombination reaction, viral genomic DNA was transfected into BHK (Baby Hamster Kidney fibroblast) cells for the production of recombinant virus. Virus was harvested from transfected BHK cells, then used to infect Vero (African Green Monkey (Chlorocebus sp.) kidney epithelial) cells. Individual plaques from infected Vero cells were collected and passaged to new Vero cells. This process was repeated for a total of four rounds of plaque isolation. Virus stocks were then generated by infection of ¨3.2 x 107 BHK cells with ¨3.2 x 105 plaque forming units (PFU) of virus and culturing for three days. After three days, supernatants were spun twice at 2,100g to pellet cells and debris. After pelleting the cells, the supernatant containing virus was spun at 17,200g to pellet virus.
Virus was resuspended, filtered, and titered on Vero cells. Viral seed stocks and research stocks were produced from purified aROR1/aCD3 BspAb viruses SepGI-189 (oil aROR1 scFv), SepGI-201 (s10 aROR1 scFv), and SepGI-203 (j1v1011 aROR1 scFv).
Table 1. ROR1 antibodies, scFvs, Bispecific antibodies, and Engineered viruses.
ROR1 VH VL aROR1 aROR1/aCD3 Oncolytic HSV
antibody domain domain scFv BspAbBspAb expressing (precursor) aROR1/aCD3 SEQ ID SEQ ID SEQ ID SEQ ID BspAb NO NO NO NO
oil 1 5 9 36 SepGI-slO 10 14 18 38 SepGI-j1v1011 19 23 27 40 SepGI-Example 2. Production of Virus-Free Conditioned Media (VFCM) from HSVs expressing citROR1icitCD3 BspAb constructs.
[00162] To generate virus-free conditioned medium (VFCM) from aROR1/a-CD3BspAb viruses SepGI-189, SepGI-201, and SepGI-203, 12-well plates were seeded with 3 x 105 A431 cells, or, in separate plates, HepG2 cells, in 1 mL of medium at 37 C, 5% CO2. The next day, the A431 cells and HepG2 cells were infected with recombinant HSVs at MOI
(Multiplicity of Infection) 0.5 and incubated for 3 days in 1.25 mL of medium.
After 3 days, cell supernatants were removed and filtered through 0.1j.tm membranes (Pall Acrodisc Syringe filter part #4611) to remove virus. The VFCMs were then aliquoted and stored at -80 'C. VFCMs of SepGI-Null, the Seprehveck HSV vector not including an exogenous transgene, was also prepared as a control.
Example 3. Detection of (FROR1icr,CD3 bispecific antibodies in VFCMs.

[00163] A 96-well plate was coated with 50 .it/well of recombinant human RORI
Fc fusion protein at 2 pg/mL (R&D Systems, Cat No. 9490-RO-050). The plate was then sealed and incubated overnight at 4 C. The next day, the plate was washed with 150 pi/well of wash buffer (Dulbecco's phosphate-buffered saline lx with 0.05% VN Tween20).
Non-specific binding was blocked using 80 4/well of blocking buffer (Dulbecco's phosphate-buffered saline with 2% BSA + 0.05% Tween20) and the plate was incubated at 37 C for 1 hour. After three washes, aROR1/aCD3 BspAb-containing virus-free culture medium (VFCMs) of SepGI-189, SepGI-201, or SepGI-203 as well as control VFCM (SepGI-Null) were serially diluted in blocking buffer and 50 u.L/well incubated for 2 hours at room temperature (RT) under slow shaking conditions. The plate was washed three times with washing buffer and 50 4/wel1 of anti-CD3 Hum291 anti-idiotype clone 5A2 diluted in blocking buffer (1:80 dilution) was added. The plate was incubated for I hour at 37 C. After three washings 50 4/well of goat anti-rabbit IgG HRP antibody (Abcam; Cat. No.
ab6721) diluted in blocking buffer at 1:120,000 dilution was added. The plate was incubated for 1 hour at 37 C in the dark. The plate was washed three times with washing buffer and SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution (SeraCare, Cat No. 5120-0082) was added to the wells (80 uL/well). The plate was incubated for 10-15 minutes at RT in the dark. Signal development was stopped by adding 50 pt/well of TMB
Blue STOP Solution (SeraCare, Cat No. 5150-0022), subsequently the signal was read at 450 nm (specific to SeraCare TMB BlueSTOP Solution) using TecanSpark or other devices.
Figure 2A provides a schematic of the assay. Figure 2B shows that all three bispecific constructs were expressed by engineered SepGI oncolytic viruses SepGI-189, SepGI-201, and SepGI-203 and were able to bind ROR1.
Example 4. Binding of oR0R1/uCD3 BspAbs from VFCM of virus-infected cultures to ROTH-positive tumor cells.
1001641 A549 human alveolar adenocarcinoma (non-small cell lung cancer) cells were knocked out for the RORI gene using CRISPR/Cas-9 methods. A549/ROR1-knockout (A549/ROR1-KO) cells or A549 wild-type (WT) cells were transferred into a V-bottom 96-well plate (80,000 cells per well). Preparations of virus-free culture medium (VFCMs) produced as described in Example 2 were serially diluted (1:5 to 1:3,125 dilution) in FACS
buffer (PBS IX + 2% FCS/FBS), added to the wells (100 mt/well), and incubated for I hour at room temperature with the cells. After three washes, the cells were resuspended in 100 u.L/well of monoclonal rabbit anti-CD3 Hum291 anti-idiotype antibody (clone 5A2) diluted at 10 [ig/mL in FACS buffer. The plate was covered with plate seal and incubated 1 hour at 37 C.
[00165] The cells were then resuspended in 100 [IL/well of FACS buffer containing donkey anti-rabbit-APC (Southern Biotech ; Cat No. 6441-31-31, Lot No. K2916-Z779B) diluted 1:1000 and the plate was incubated for 1 hour at 37 C in the dark.
Cells were finally resuspended in 120 [tL/well of FACS buffer and the signal was analyzed on an AttuneNxt flow cytometer. Figure 3A provides the assay format, where A549 WT cells expressing ROR1 bind the BspAb present in the VFCM which in turn is recognized by the anti-idiotypic anti-CD3 antibody. The complex is revealed using an Allophycocyanin (APC)-labelled donkey anti-rabbit antibody. No binding of A549/ROR1-K0 cells by the BspAbs present in the VFCM is expected to occur. Figure 3B provides the flow cytometry results showing that all VFCMs of viruses that included bispecific constructs contained aROR1/aCD3 bispecific antibodies that bound ROR1-expressing A549 tumor cells but failed to bind cells. The VFCM prepared from the culture of cells infected with the control virus that did not include a bispecific construct (SepGI-Null) did not contain antibodies that were able to bind the cells and anti-idiotypic CD3 antibody.
Example 5. Binding of uROR1/uCD3 bispecific antibodies from VFCM of virus-infected cultures to ROR1-positive tumor cells.
[00166] A549-WT and A549-ROR1 KO cells were stained with eFluor450 dye (Thermo Fisher Scientific; Cat. No. 50-246-096) as recommended by the manufacturer.
Purified human T cells were freshly isolated from healthy blood donor using the PAN T-Cell isolation kit, human (Miltenyi Biotec; Cat No. 130-096-535) and stained with eFluor670 dye (Thermo Fisher Scientific; Cat. No. 65-0840-85) as recommended by the manufacturer.
The cells were resuspended at 1.0E+07 cells/mL in Dulbecco's 1X phosphate-buffered saline (DPBS) at 37 C. eFluor450-labelled A549-WT or A549-ROR1 KO tumor cells were mixed with purified eFluor670-labelled T cells at a 1:1 ratio (30,000 tumor cells and 30,000 T
cells/well) in a U-bottom low adherence 96-well plate (in 100 [iL/well of complete RPMI 1640 media containing 10% FBS). Cells were centrifuged 3 min at 1,500 rpm and the supernatant removed by quickly flicking the plate. The cell pellets were resuspended in 501AL of undiluted virus-free culture medium (VFCM) containing an aCD3/aROR1 bispecific construct (SepGI-189, SepGI-201, or SepGI-203). Cells were incubated for 1 hour at 37 'C.
Subsequently, cells were fixed with 100 [IL of fixation buffer (Biolegend;
Cat. No. 420801, Lot No. B295965) added directly to the wells and the cells plus antibody were incubated for 20 min at room temperature in the dark without disrupting the cells or pipetting. Samples were immediately analyzed on the Attune NxT cytometer without washing or pipetting.
Figure 4A shows the assay design, where a BspAb that binds both ROR1 (expressed on WT
A549 cells) and CD3 (expressed on T cells) is able to bind both eFluor 450-labeled WT A549 cells and eFluor 670-labeled T cells (but is not able to bind A549/ROR1-K0 cells).
[00167] Figure 4B shows the fluorescence quadrants where labeled T cells (alone) and labeled WT A549 cells (alone) are found after flow cytomety. The rightmost plot shows that when the cells are mixed in the presence of VFCM, cell appear in a new region of the plot indicating that both fluorophores have spatially come in close contact. The graph shows the percent interaction of T cells with WT A549 cells and A549/ROR1-K0 cells for VFCMs made from cultures infected with the SepGI-189 virus, the SepGI-201 virus, and the SepGI-203 virus, each of which includes a aROR1/aCD3 BspAb construct. In each case, the interaction of WT A549 cells and CD3 cells is dramatically higher than the interaction of A549/ROR1-K0 cells and CD3 cells, showing the all three bispecific antibodies are able to simultaneously bind to tumor and T cells in an antigen-specific manner.
Example 6. Functional activity uROR1/uCD3 bispecific antibodies of VFCMs.
[00168] To test the ability of a bispecific aROR1/aCD3 antibody to induce signaling in T
cells, an assay using Jurkat cells having a luciferase gene under the control of a NFAT
response element (Jurkat-NFAT-Luc) was used. Figure 5A depicts the assay set-up where BspAb bound to ROR1-expressing A549 cells also binds CD3 on Jurkat cells, resulting in signaling that leads to luciferase expression and a luminescent signal.
A549/ROR1-K0 cells that do not bind the aROR1/aCD3 BspAb do not stimulate Jurkat cell signaling.
[00169] To perform the assay, 20,000 A549-WT or A549-ROR1 KO cells were plated in a white opaque, flat-bottom 96-well assay plates (Corning Cat.# 3917) in 100 [IL
of complete RPMI-1640 (RPMI-1640 containing 10% FCS). The plate was spun 1 mm at 1,500 rpm and incubated overnight at 37 C to let the cells adhere. The plate was then spun for 3 mm at 1,500 rpm and the supernatant was discarded by quickly flicking the plate.
Jurkat cells expressing luciferase under the control of a NFAT response element (Jurkat-NFAT-Luc) were then plated in the wells (30,000 cells in 50 [IL of complete RPMI-1640 medium/well).
Cell activation was induced by adding 50 pt/well of either aROR1/aCD3 (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 in separated wells at 2 pg/mL as a positive control for T cell activation. The plate was incubated for 5 hours at 37 C in a humidified cell incubator. Luminescent signal was revealed by adding 100 pt/well of Bio-Glo Luciferase Assay substrate (Promega; Cat No. G7940; Lot. No. 0000422404) as recommended by manufacturer and the plate was incubated at room temperature for 5 min in the dark under slow shaking conditions. The luminescent signal was read with the TECAN Device (integration time: 500ms). Figure 5B shows that all three VFCMs induced T cell activation, with SepGI-201 and SepGI-203 VFCMs inducing potent T cell activation in a ROR1-dependent manner.
Example 7. Cytotoxicity assays using VFCMs containing uROR1/uCD3 bispecific antibodies.
[00170] To test the killing of ROR1-expressing tumor cells by T cells in the presence of the aROR1/aCD3 bispecific antibodies, cytotoxicity assays were performed as follows. On day 0, 10,000 A549-FLuc WT and A549-FLuc ROR1 KO cells (target cells) were plated in 100 [IL of complete RPMI1640 (RPMI1640 supplemented with 10% FCS) in white opaque, flat-bottom 96-well assay plates (Corning Cat.# 3917). The plate was spun 1 min at 1,500 rpm and incubated overnight at 37 C to let the cells adhere.
[00171] On day 1, human peripheral blood mononuclear cells (hPBMCs) were isolated from human healthy whole blood and human T cells were isolated from hPBMCs using a pan T cell isolation kit (Miltenyi Biotec; Cat. no. 130-096-535, lot 519115439) as recommended by the manufacturer.
[00172] The supernatants from the 96-well plate containing target cells were removed by quickly flicking the plate, and VFCMs containing a-ROR1/a-CD3 bispecific antibodies were diluted in complete RPM11640 and added to the target cells at 100 L/well.
Subsequently, 100 [it/well of purified human T cells (effector cells) were added (5,000 cells/well) on top of the target cells to reach an E:T ratio of 0.5:1. As a control, some wells did not receive effector cells. Cells were gently mixed, spun for 1 min at 1,500 rpm and incubated for 3 days at 37 C. On day 4, The supernatants (100 pt/well) were collected to measure IF1\17 expression levels for each condition using the proinflammatory panel 1 (human) kit from Meso Scale Discovery (MSD; Cat. No. K15049D) by following the manufacturer's recommendations.
The killing activity was evaluated by measuring the luminescent signal which was revealed by adding 100 4/well of Bio-Gle Luciferase Assay substrate (Promega; Cat No.
G7940;
Lot. No. 0000422404) as recommended by the manufacturer and incubated at room temperature for 8 min in the dark under slow shaking conditions. Luminescent signal was read with the TECAN Device (integration time: 500ms). Percent killing of the samples was calculated as follows: 100 ¨ (IlLuminescence sample / Baseline Luminescence no VFC'M control]) X
100.

[00173] The results provided in Figure 6 show that VFCMs of cells infected with SepGI-189 and SepGI-201 were able to stimulate killing of ROR1 -expressing tumor cells by T cells, and that this efficient killing was specific for tumor cells expressing ROR1.
T cells co-cultured with target cells in the presence of the aROR1/aCD3 bispecific antibodies also secreted significant amounts of interferon gamma.
Example 8. Cytotoxicity assays including VFCMs containing uROR1/uCD3 bispecific antibodies using tumor lines with different levels of ROR1 expression.
[00174] ROR1 expression 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 washed twice using 170 4/well of FACS buffer (PBS 1X + 2% FCS/FBS + 0.1%
sodium azide). A purified human anti-human ROR1 antibody was diluted in FACS buffer at various concentrations (ranging from 10 to 0.00061 Rg/mL; dilution 1:4), then cells were resuspended in 100 4/well of diluted antibody and incubated for 30 min at 4 C.
After 2 washes in 170 jiL/well of FACS buffer, cells were incubated with an AF647-conjugated goat anti-human IgG secondary antibody (Southern Biotech; Cat. no. 2040-31, lot.
K471X873C;
dilution 1:2,000 in FACS buffer) at 80 4/well for 20 mm at 4 C. Cell pellets were washed twice, and subsequently resuspended with 120 4 of fixation buffer (Biolegend;
Cat No.
420801, Lot No. B306498) and incubated for 15 min at room temperature in the dark. Then, cells were centrifuged at 1,500 rpm for 2 min and the supernatant removed by quickly flicking the plate. Cells were washed twice, resuspended in 150 4/well of FACS
buffer and acquired by flow cytometry on the Attune NxT. Data were analyzed by using FlowJo v10.
Figure 7A shows that of the human tumor cell lines, A549 (alveolar adenocarcinoma) has the highest level of ROR1 expression, and HepG2 (liver cancer) express little ROR1, with the detected labeling with ROR1 antibody being comparable to that of A549-ROR1 knockout cells. MCF-7 (breast cancer) cells expressed an intermediate level of ROR1.
[00175] To perform the killing assay, human peripheral blood mononuclear cells (hPBMCs) were isolated from human healthy whole blood, then human T cells were isolated from hPBMCs using the EasySep human T cell Isolation kit (StemCell Technology;
Cat. No.
17951, lot 1000024139) as recommended by the manufacturer. A549-Fluc WT, A459-Fluc/ROR1 KO, MCF-7-FLuc and HepG2-Fluc target cells were plated at 10,000 cells/well in 100 RI. of complete culture medium in white opaque, flat-bottom 96-well assay plates (Corning Cat.# 3917). The plate was spun 1 min at 1,500 rpm and incubated overnight at 37 C to let the cells adhere. The supernatants from the 96-well plate containing target cells were removed by quickly flicking the plate. Purified effector T cells (1001dt/well) were plated on top of target cells at a 2:1 E:T ratio. As controls, some wells did not receive effector cells.
VFCM of SepGI-201-infected cultures or SepGI-Null-infected cultures were diluted in complete RPMI1640 and added to the cells at 100 ut/well. The SepGI-201 virus includes the aROR1/aCD3 BspAb construct and the SepGI-Null virus does not include a BspAb construct. Cells were gently mixed, spun for 1 min at 1,500 rpm and incubated for 3 days at 37 'V, after which the supernatants (100 pi/well) were collected. IFNy levels present in the supernatants were measured using the proinflammatory panel 1 (human) kit from Meso Scale Discovery (MSD; Cat. No. K15049D) by following the manufacturer's recommendations.
Killing activity was evaluated by measuring the luminescent signal from the wells by adding 100 4/well of Bio-Glo Luciferase Assay substrate (Promega; Cat No. G7940;
Lot. No.
0000422404) as recommended by the manufacturer and incubating at room temperature for 5 min in the dark under slow shaking conditions. The luminescent signal was read with the TECAN Device (integration time: 500ms). Percent killing of the samples was calculated as follows: 100 ¨ (ILuminescence sample / Baseline Luminescence no VF CM
control]) X 100. Figure 7B demonstrates that while A549 and MCF-7 cells were killed by T cells in the presence of SepGI-201 VCFM, HepG2 cells were preserved (due to low ROR1 expression) even if T
cells were activated at high VFCM concentration, as demonstrated by increased IFNy expression.
Example 9. Killing activity of oncolytic viruses SepGI-189, SepGI-201, and SepGI-203 expressing bispecific antibodies.
[00176] Figure 8A provides the experimental plan for evaluating killing of A549 tumor cells by oncolytic viruses SepGI-189, SepGI-201, and SepGI-203, expressing the "oil"
aROR1/aCD3, "s10" aROR1/aCD3 and "j1v1011" aROR1/aCD3 BspAb constructs, respectively (Table 1). On day 0, A549-Fluc WT and A549-Fluc ROR1 KO target cells were plated at 10,000 cells/well in 100 pt of complete RPMI-1640 + 10% FCS in white opaque, flat-bottom 96-well assay plates (Corning Cat.# 3917). The plate was spun 1 min at 1,500 rpm and incubated overnight at 37 C to let the cells adhere. On day 1, the target cells were infected with either an ctRORI/aCD3 virus (SepGI-189, SepGI-201, or SepGI-203, see Table 1) or the negative control SepGI-Null virus at multiplicities 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. no. 130-096-535, lot 519115439) as recommended by the manufacturer. The supernatants were removed from target cells by quickly flicking the 96-well plate and 20,000 effector T cells plated in 100 pt/well to reach an E:T ratio of 2:1. Cells were gently mixed, spun for 1 min at 1,500 rpm, and incubated for 4 days at 37 'C. On day 6, the killing activity was evaluated by measuring the luminescent signal using 100 uL/well of Bio-Glo Luciferase Assay substrate (Promega; Cat No. G7940;
Lot. No. 0000422404) as recommended by the manufacturer and incubated at room temperature for 5 min in the dark under slow shaking conditions. Luminescent signal was read with the TECAN Device (integration time: 500ms). Percent killing of the samples was calculated as follows: 100 ¨ (Luminescence sample / Baseline Luminescence no virus]) X 100.
[00177] Figure 8B shows that tumor cells infected with each of the BspAb-expressing viruses at MOIs as low as 0.11 were killed at significantly percentages than tumor cells infected with the SepGI-null virus. Infection of tumor cells with SepGI-201 led to significantly higher killing of tumor cells at an MOI of 0.04, while infection with of tumor cells with SepGI-203 led to significantly higher killing of tumor cells at an MOI of 0.01. The same effect was not seen when ROR1 knockout tumor cells were infected with viruses and used as targets. In this case, only infection with the SepGI-203 virus led to significantly higher killing demonstrating that SepGI-203 showed some degree of non-specific killing at MOI higher than 0.11. All together these data showedthat oncolytic activity combined with aROR1/aCD3 BspAb significantly increased anti-tumor activity in an antigen-specific dependent manner.
Example 10. Mouse cross-reactivity of slO and j1v1011 monoclonal antibodies.
[00178] To test whether the slO (R06D8-s10) and j1v1011 (RO6D8-j1v1011) monoclonal antibodies used in engineering the aROR1/aCD3 bispecific antibodies recognized mouse ROR1 in addition to human ROR1, the assay depicted in Figure 9A was employed.

well plate was coated with 50 uL/well of recombinant mouse ROR1 IgG2-Fc fusion protein at 2 ug/mL (R&D Systems, Cat No. 9910-RO-050, Lot No. DIWM0120121), the plate was then sealed and incubated overnight at 4 C. The next day, the plate was washed with 150 pL/well of wash buffer (Dulbecco's phosphate-buffered saline lx with 0.05% VN
Tween20). Non-specific binding was blocked by using 80 iL/well of blocking buffer (Dulbecco's phosphate-buffered saline with 2% BSA + 0.05% Tween20) and the plate was incubated at 37 C for 1 hour. After three washes, two anti- human ROR1 antibodies (s10 and j1v1011) were serially diluted in blocking buffer (80 uL/well) and incubated for 2 hours at room temperature (RT) with slow shaking. The plate was washed thrice with wash buffer and then 80 L/well of secondary HRP-labelled goat anti-human IgG Fc (SouthernBiotech; Cat No. 2081-05; Lot No. L5311-TE40) diluted in blocking buffer (1:2,000 dilution) was added. The plate was incubated for 1 hour at 37 C in the dark. The plate was washed thrice with washing buffer and SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution (SeraCare, Cat No. 5120-0082) was added to the wells (80 [it/well). The plate was incubated for 10-15 minutes at RT in the dark. The signal development was stopped by adding 50 rat/well of TMB Blue STOP Solution (SeraCare, Cat No. 5150-0022), and subsequently the signal was read at 450 nm (specific to SeraCare TMB BlueSTOP Solution) using TecanSpark or other devices. Figure 9B shows that both anti-RORI antibodies used to generate the BspAb constructs of SepGI-201 and SepGI-203 exhibit mouse-cross-reactivity.
Example 11. In vivo study of tumor treatment with virus expressing BspAb.
[00179] Figure 10A provides a diagram of the inoculation and treatment schedule of mice used to test the effectiveness of oncolytic viruses expressing aROR1/aCD3 bispecific antibodies. On day ¨6 (D-6), Female NSG-Tg(Hu-IL-15) mice (6 weeks of age) were injected intraperitoneally (I.P.) with 1.0E+07 freshly purified human peripheral blood mononuclear cells (PBMCs) in Dulbecco's phosphate-buffered saline (DPBS) lx. On day 0 (DO), mice were injected subcutaneously (S.C.) in the right flank with 5.0E+06 A549-WT
tumor cells diluted in 100 [IL of DPBS 1X. On day 6 (D6), mice were randomized into four groups (three 'viral treatment' groups and one 'no viral treatment' control group) and viral treatments were initiated: 50 .it/mouse/injection of either SepG1-189, SepG1-201, SepGI-Null, or no virus was delivered peri-tumorally (P.T.) on days 6, 10 and 12. Tumor growth and body weight were monitored twice weekly. Tumor volume was measured using a caliper and calculated using the formula V=(LengthxWidth2)/2. The study was terminated on day 31 and percent tumor growth inhibition (TGI) was calculated as follows: [1 ¨ (Relative tumor volume of the treated group)/(Relative tumor volume of the control group)] x 100.
[00180] Figures 10B and 10C provide the tumor volumes and calculated tumor growth inhibition (TGI) for the treatment groups and non-treatment group and Figure 10D provides mouse body weights over the course of the experiment. Treatment with viruses expressing aROR1/aCD3 bispecific antibodies (SepGI-189 and SepGI-201) led to greater inhibition of tumor growth than either no treatment or treatment with a virus (SepGI-Null) that did not express an aROR1/aCD3 BspAb.
Example 12. Constructs for expressing ROR1/CD3 Bispecific antibodies together with additional gene encoding IL-12 or IL-12 plus an anti-VEGFR antibody.
1001811 Additional constructs were made for the synthesis of HSVs encoding a CD3 bispecific antibody and in addition, the cytokine IL-12. The SepGI-216 construct (a double gene construct, Figure 11A) included the aROR1(s10)-aCD3 bispecific antibody (SEQ ID NO:18) under the control of the EF1a/HTLV promoter (SEQ ID NO :41) and a 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 encoded a single polypeptide (SEQ ID NO:47) encompassing both the p40 and p35 subunits of IL-12 connected by a 2x elastin linker (SEQ
ID NO:66). The SepGI-212 construct (a triple gene construct, Figure 11B) included a gene encoding the aROR1(s10)-aCD3 bispecific antibody (SEQ ID NO:37) under the control of the EFla/HTLV promoter (SEQ ID NO:41) and a gene encoding an anti-VEGFR2 scFv linked to an Fcl region (SEQ ID NO:50) and the human IL-12 gene (SEQ ID
NO:46), where the sequence encoding the aVEGFR2 scFv-Fcl (SEQ ID NO:48) and the sequence encoding human IL-12 (SEQ ID NO:46) were separated by a sequence encoding a T2A self-cleavage peptide (SEQ ID NO:51) and the continuous open reading frame encompassing sequences encoding the human IL-12 polypeptide and the VEGFR2 scFv-Fcl polypeptide was under the control of the CMV promoter (SEQ ID NO:42) (see, Figure 11B).
1001821 In addition, for use as controls, analogous constructs were designed in which the gene encoding the aROR1(s10)-aCD3 bispecific antibody was replaced by a gene encoding a bispecific antibody that included an scFv that bound the F protein of Respiratory Syncyti al Virus (SEQ ID NO:43) and CD3 (referred to herein as the -aRSV-aCD3 bispecific antibody"). Cloning of these constructs and production and isolation of recombinant viruses was performed essentially as set forth in Example 1.
Table 2. SepGI HSV constructs.
Virus Transgenes SepGI-Null none SepGI-201 aROR1(s10)-aCD3 SepGI-207 aRSV-aCD3 SepGI-212 aROR1(s10)-aCD3; aVEGFR2-Fc; hulL-12 SepGI-214 aRSV(s10)-aCD3; aVEGFR2-Fc; huIL-12 SepGI-216 aROR1(s10)-aCD3; huIL-12 SepGI-218 aRSV(s10)-aCD3, huIL-12 [00183] VFCMs produced from the viruses (see Example 2) was tested to assess by ELISA for the expression of the transgenes essentially as described in Example 3, where the wells of the plates were coated with either ROR1, the RSV protein, or human VEGFR.
[00184] The results of the ELISAs are provide in Figures 12A, B, and C. The first graph (Figure 12A) shows that all three viruses that included the gene encoding the RSV protein antibody (SepGI-207, SepGI-214, and SepGI-218) did produce the RSV antibody, whereas none of the other viruses (lacking the RSV protein antibody) did. Figure 12B
shows that viruses SepGI-201, SepGI-216, and SepGI-212 all expressed the ROR1 antibody, as expected, whereas the control viruses lacking the gene encoding the aROR1(s10)-aCD3 bispecific antibody did not. Figure 12C shows the results of the ELISA used to detect IL-12.
In this case, VFCMs from cells infected with SepGI-212, SepGI-216, and SepGI-218 were all found to contain IL-12 protein, whereas the two isolates of cells infected with SepGI-214 did not. (Subsequent isolates of SepGI-214 were later found to produce IL-12.) As expected, IL-12 was not detected in VFCMs of uninfected cultures or cultures infected with the SepGI
Null virus or the SepGI-207 virus.
[00185] The results of an ELISA for detecting the VEGFR2 scFv antibody are shown in Figure 13, where the wells of the 96 well ELISA plates were coated with recombinant human VEGFR2 (VEGFR2/KDR Protein (ECD, His Tag) (Sino Biological). After washing the antigen-coated wells, VFCMs were 8-fold serially diluted in blocking buffer and added at 50 uL/well and the plate was incubated for 2 h at room temperature on a shaker.
The plate was washed 3X with wash buffer and 50 L/well of Goat anti Human IgG (H+L) Secondary Antibody, HRP (diluted 1:5,000x in blocking buffer) was added (Invitrogen) and the plate was incubated for 1 h at 37 C. After the plate was washed 3X with wash buffer, the signal was detected by using 50 pt/well of SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate Solution (Cat No. 5120-0082, SeraCare). The plate was incubated 10-12 min at room temperature in the dark. Then 50 L/well of TMB BlueSTOP Solution (Cat No.
5150-0022, SeraCare) was added and the absorbance read at 450 nm (specific to SeraCare TMB BlueSTOP Solution) using TecanSpark. The graph of Figure 13 shows that the VFCM
of one isolate of the triple gene virus SepGI-212 demonstrated expression of the uVEGFR2-Fc antibody, whereas there was no binding of any of the VCFMs of viruses that did not include the aVEGFR2-Fc antibody gene (SepGI-Null, SepGI-201, SepGI-207, SepGI-216, and SepGI-218).
Example 13. IL-12 Activity Assays.
[00186] Assays for the activity of IL-12 were performed essentially as described in Example 9, where the VFCMs of cells infected with SepGI-Null, SepGI-201, SepGI-207, SepGI-212, SepGI-214, SepGI-216, and SepG1-218 were tested in luciferase-based assays. A
cell-based assay was used in which cells having 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 the recombinant HSVs. Promega Corporations One-Glo Luciferase system was used for detection.
[00187] Briefly, the assay was performed by adding diluted VFCM
from uninfected cells or cells infected with various HSVs to the wells of a 96 well plate. The lysates of infected cell cultures (VFCMs) were produced as described in Example 2. A dilution series of recombinant IL-12 (R&D Systems) was added to additional wells to generate a standard curve. The IL-12 reporter cells were used essentially according to the manufacturer's instructions. 40K iLite Cells were thawed, diluted, and 40 1 was added to each well of a 96-well plate. 40 1 of a dilution series of the VFCMs was then added to the assay wells, the contents of the wells were mixed, and the plate was incubated for five hours at 37 C, 5%
CO2. Recombinant IL-12 was added in dilution series to separate wells for generating a standard curve. The One-Glo luciferase reagent (Promega Corp., Madison, WI) was then added to each well (40 L) and after 10 min at room temperature, firefly luciferase luminescence was measured using a Tecan Spark plate reader. The results are shown in Figure 14 which provides a graph of the luminescence from assays using uninfected cell conditioned media, conditioned media from cells infected with a virus that did not include exogenous transgenes (SepGI-Null), and conditioned media from cells infected with the IL12 gene-containing viruses SepGI-201 and SepGI-207 (no IL12 gene), SepGI-212 and SepGI-214 (triple gene viruses with IL-12 gene). and SepGI-216 and SepGI-216 (double gene viruses with IL-12 gene). Notably, all cells infected with viruses that included the IL-12 gene expressed functional IL-12, with the exception of isolates of the triple gene virus SepGI-214 that also did not show production of the IL-12 protein in the ELISA (Example 12).
Example 14. Cell-Cell interaction assays.
1001881 To assess the ability of the aROR1-aCD3 bispecific antibodies encoded by the engineered HSVs to conjugate target ROR1-expressing tumor cells and T cells, mouse tumor cells and human T cells were separately labeled with fluorophores. Hepa 1-6 cells and A549 cells, both of which express ROR1, were labeled with eFluor 450 (ThermoFisher) (Figure 15B) using and human T cells isolated from PBMCs were pre-labled with eFluor 670 (Figure 15C) and cell-cell interactions were assayed and analyzed by flow cytometry essentially as described in Example 5. Figure 15D shows an example of the flow cytometry results, where conjugated cells (fluorescing at both wavelengths) are seen in the upper right quadrant of the plot.

[00189] The results of flow cytometric assays for aROR1-aCD3 bispecific antibody-mediated conjugation of T cells with ROR1-expressing tumor cells is presented graphically in Figure 16A, B, and C. Figure 16A shows, from left to right, the percentage of Hepa 1-6 cells, A549 wild type cells, and A549 ROR1 knockout cells that were conjugated to T cells after co-incubation in the presence of SepGI-218 VFCM which expresses a construct that encodes an aRSV-aCD3 bispecific antibody as well as IL-12. Figure 16B shows, from left to right, the percentage of Hepa 1-6 cells and then A549 wild type cells after co-incubation in the presence of SepGI-201 VFCM which expresses a construct that encodes an aROR1-aCD3 bispecific antibody. Figure 16C shows, from left to right, the percentage of Hepa 1-6 cells and then A549 wild type cells after co-incubation in the presence of SepGI-216 VFCM
which expresses a construct that encodes an aROR1-aCD3 bispecific antibody as well as IL-12. Minmal cell-cell interaction is observed in the presence of the SROR1+
Tumor cell- T
cell interaction is observed in the presence of VFCM of SepGI-201-infected cells and SepGI-216-infected cells, with no significant differences observed between SepGI-201-infected cells and SepGI-216-infected cells.
Example 15. T-Cell Activation Assays.
[00190] To determine the effect of the aROR1-aCD3 bispecific antibodies on T
cell activation, assays were performed in which ROR1-expressing tumor cells were incubated with T cells in the presence of VFCMs of cells infected with viruses encoding the aROR1-aCD3 bispecific antibodies, after which activation markers on the surfaces of the T cells were assessed. Briefly, wild type A549 cells, or as controls, ROR1 knockout A549 cells, were plated in the wells of 96 well plates at 104 cells per well. The next day, freshly isolated CD3+
T cells, stained with CFSE, were added to the wells at E:T ratios of 10:1 or 5:1. VFCM was added to the wells at a 1,000 fold dilution, or, as positive controls, CD3/CD28 beads were added to the wells (bead:cell ratio of 1:20). One, two, and three days later supernatant was removed for staining of T cells for the expression of activation markers and analysis by flow cytometry.
[00191] Figure 17A-D provide the results of assays with ROR1 knockout A549 cells as targets. Figure 17A shows that the viability of the CD3+ T cells on days 1, 2, and 3 of the assay was close to 100% regardless of whether the cells were cultured with VFCM of uninfected cultures (first two bars) or with VFCM of cells infected with the SepGI-Null virus (second two bars), the SepGI-207 virus (second two bars), the SepGI-201 virus (third two bars), or CD3/CD28 beads (fourth two bars). Figure 17B provides the CD3+ CD4+
cell count for each assay group on successive days of the assay. Figure 17C
provides the percentage of CD25+ cells for each assay group on successive days of the assay based on flow cytometry, and Figure 17D provides the percentage of CD69+ cells for each assay group on successive days of the assay. Although CD3/CD38 beads resulted in activation of the T cells as evidenced by increased expression of both CD25 and CD69 over the course of the assay, when ROR1 knockout cells were used as targets, no activation of the T cells was observed as assessed by expression of CD25 and CD69, regardless of the presence of VFCM.
[00192] Figure 17E-H provide the results of assays with wild type A549 cells that express ROR1 as targets. Figure 17E shows that the viability of the CD3+ T cells on days 1, 2, and 3 of the assay was close to 100% regardless of whether the cells were cultured with VFCM of uninfected cultures (first two bars) or with VFCM of cells infected with the SepGI-Null virus (second two bars), the SepGI-207 virus (second two bars), the SepGI-201 virus (third two bars), or CD3/CD28 beads (fourth two bars). Figure 17F provides the CD3+ CD4+
cell count for each assay group on successive days of the assay. Figure 17G
provides the percentage of CD25+ cells for each assay group on successive days of the assay based on flow cytometry, and Figure 17H provides the percentage of CD69+ cells for each assay group on successive days of the assay. Notably, the presence of VFCM of cultures infected with the SepGI-201 virus that was engineered to express the aROR1-aCD3 bispecific antibody resulted in expression of both CD25 and CD69 by the T cells in the co-culture. This induced expression was not observed for co-cultures that instead included the VFCM of SepGI-207 infected cells, with the VFCM of SepGI-207 infected cells, or with the VFCM of uninfected cells. Thus, using target cells that expressed ROR1, the activation of T cells in co-cultures could be attributed to the presence of the aROR1-aCD3 bispecific antibody, which can engage the T cells leading to their activation.
Example 16. T cell proliferation/activation assays.
[00193] An additional cell culture assay was performed with single and double gene expressing viruses. In these assays A549 wild type or A549 ROR1 knockout cells were plated in the 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 10:1 and 5:1 effector:target ratios, and VFCMs at 1:1,000 dilution were added to the wells. The VFCMs were of cells infected with SepGI-207 (aRSV-aCD3 bispecific antibody gene), SepGI-201 (aROR1-aCD3 bispecific antibody gene), SepGI-216 (aROR1-aCD3 bispecific antibody gene plus IL-12 gene), and SepGI-218 (aROR1-aCD3 bispecific antibody gene plus IL-12 gene). The plates were incubated for 3 days, with flow cytometry performed after 1, 2, or 3 days to determine cell proliferation by the percentage of CTV+ T cells. Figure 18 shows the results of assays at a 5:1 effector to target ratio, where specific T cell proliferation was observed only for ROR+
target cells and only when the aROR1-aCD3 bispecific antibody-containing VFCMs of SepGI-201 and SepGI-216 infected cultures were included in the cultures.
Example 17. Luciferase-based killing assay using VFCMs of cells infected with single, double, and triple gene HSVs engineered to express aROR1-uCD3 bispecific antibodies.
[00194] Assays were performed to assess the effects of VFCMs of cells infected with HSVs engineered to express aROR1-aCD3 bispecific antibodies on the killing of ROR1+
tumor cells by T cells. For these assays, target cells (A549 wild type cells or A549 ROR1 knockout cells for use as controls) were labeled by transducing the cells with a retrovirus for expressing GFP and firefly luciferasel. The luciferase-expressing target cells were plated at 104 cells per well in 100 1RPMI-1640+10% FCS in 96 well plates and cultured for two days at 37 C. Freshly isolated human T cells freshly isolated from PBMCs were then added to the wells at a ratio of 0.5:1 and VFCMs at dilutions of 1,000 or 1:8,000 were added to each assay well. The plates were incubated for four days at 37 C, after which the number of luceriferase-expressing cells was assessed by adding 80 ill of Bio-Glo Luciferase Assay reagent (Promega), incubating the plate in the dark for 5 min, and reading luminescence with a TECAN device (integration time, 500 ms). Figure 19A shows that in the absence of T cells (effectors) the number of A549 wild type cells is close to 107 regardless of the presence or type of VCFM added to the culture. In the presence of T cells however, a reduction in A549 wild type target cells is evident in cultures that included VFCMs of viruses engineered to express the aROR1-aCD3 bispecific antibody: SepGI-201, SepGI-212, and SepGI-216 (see Table 2). Killing of target cells was not observed in cultures that included VFCMs of the SepGI-207, SepGI-214, and SepGI218 viruses that were not engineered to express the aROR1-aCD3 bispecific antibody, as also seen in the graph providing the percentage of killing, shown in Figure 19C. Figures 19B and 1911 provide the results when knockout A549 target cells were used, demonstrating lack of killing of cells that did not express ROR1 by the T cell effectors regardless of the VCFM (or bispecific antibody) in the co-culture.
Example 18. Xcelligence Killing Assay using 549 WT cells, with Single, Double, Triple gene expressors (VFCMs).

[00195] Killing assays were also performed using the xCELLigence Real Time Cell Analyzer (Acea Biosciences, San Diego, CA). For these experiments, A549 wild type and A549 ROR1 knockout cells were seeded into the wells of 96 well E-plates (Acea Biosciences) at 10,000 cells/well in 50 n1 of RPMI-1640+10% FCS. T cells were added at a 0.5:1 effector to target ratio, and 1:1,000 dilutions of VFCMs of cell cultures infected with the HSVs SepGI-Null, SepGI-207, SepGI-212, SepGI-216, and SepGI-218 were added. The plates were read continously for three days. Figure 20 shows that assays that included VFCMs of HSVs that did not include aROR1-aCD3 bispecific antibody constructs:
SepGI-207, SepGI-214, SepGI-218, and SepGI-123 (IL-12 gene only), proliferated to essentially the same extent and with the same pattern as cultures that lacked VFCM altogether.
On the other hand, assays that included VFCMs of HSVs engineered to express aROR1-aCD3 bispecific antibody constructs: SepGI-201, SepGI-212, and SepGI-216, demonstrated reduced proliferation, indicating killing of the ROR1+ target cells in these cultures.
Figure 21 shows the results of parallel assay in which the target cells were ROR1 knockout cells. In this case no impairment of proliferation was observed.
Example 19. In vivo study of anti-tumor activity of SepGI-201 VFCM in NOD/Scid pseudo-humanized mouse model.
[00196] A study was designed to assess the effect of treating of tumor-bearing NOD/Scid pseudo-humanized mice with VFCM of cells infected with the SepGI-201 HSV
engineered to express an aROR1-aCD3 bispecific antibody. As controls, some tumor-bearing mice are treated with VFCM of cells infected with the SepGI-207 HSV engineered to express an aRSV-aCD3 bispecific antibody. Six groups of eight mice are established with the treatment regimens shown in Table 3.
Table 3. Groups of mice for VFCM treatment study.
Group Tumor Cells PBMCs VFCM
1 4 x 106 A549 wt 4 x 106 hPBMCs None 2 4 x 106 A549 wt 4 x 106 hPBMCs aRSV-aCD3 (SepGI-207) 3 4 x 106 A549 wt 4 x 106 hPBMCs aROR1-aCD3 (SepGI-201) 4 4 x 106 A549 ROR1 KO 4 x 106 hPBMCs None 4 x 106 A549 ROR1 KO 4 x 106 hPBMCs aRSV-aCD3 (SepGI-207) 6 4 x 106 A549 ROR1 KO 4 x 106 hPBMCs aROR1-aCD3 (SepGI-201) [00197] Tumor cells, either A549 wild type or A549 ROR1 knockout, are subcutaneously coinjected with human PBMCs in all mice. Four weeks later, treatments begin, in which mice of groups 2-6 are injected peri-tumorally with 50 1 of VFCM every four to five days for a total of five treatments. Tumor growth and body weight are monitored twice weekly. Tumor volume is measured using a caliper and on termination of the study at approximately 9 weeks tumor growth inhibition (TGI) is calculated as follows: [1 ¨ (Relative tumor volume of the treated group)/(Relative tumor volume of the control group)] x 100.
Example 20. In vivo study of anti-tumor activity of SepGI-201 VFCM in NSG-132m KO
pseudo-humanized mouse model.
[00198] A study was designed to assess the effect of treating of tumor-bearing NSG-B2m knockout pseudo-humanized mice with the SepGI-201 HSV engineered to express an aROR1-aCD3 bispecific antibody. As controls some mouse groups are treated with the SepGI-207 HSV engineered to express an aRSV-aCD3 bispecific antibody. Six groups of eight mice are established with the treatment shown in Table 4.
Table 4. Groups of mice for VFCM treatment study.
Group Tumor Cells PBMCs Virus 1 5 x 106 A549 wt 5 x 106 hPBMCs None 2 5 x 106 A549 wt 5 x 106 hPBMCs SepGI-207 (aRSV-aCD3) 3 5 x 106 A549 wt 5 x 106 hPBMCs SepGI-201 (aROR1-aCD3) 4 5 x 106 A549 ROR1 KO 5 x 106 hPBMCs SepGI-207 (aRSV-aCD3) 5 x 106 A549 ROR1 KO 5 x 106 hPBMCs SepGI-201 (aROR1-aCD3) [00199] Tumor cells, either A549 wild type or A549 ROR1 knockout, are subcutaneously coinjected with human PBMCs in all mice. Four weeks later, treatments begin, in which mice of groups 2-5 are injected peri-tumorally with 50 1 of oncolytic virus every four to five days for a total of three treatments. Tumor growth and body weight ae monitored twice weekly.
Tumor volume is measured using a caliper and on termination of the study at approximately 9 weeks tumor growth inhibition (TGI) is calculated as follows: [1 ¨ (Relative tumor volume of the treated group)/(Relative tumor volume of the control group)] x 100.

SEQUENCES
SEQ ID NO:1 Protein Artificial oil anti-ROR1 antibody Heavy Chain Variable Region QVQLVQSGAEVKKPGASVKVSCKAS GYT FTNYYMHWVRQAPGQGLEWMGI INPTSGRTSYAQKFQGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSSWYSGWYFDLWGQCTTVTVSS
SEQ ID NO:2 Protein Artificial oil anti-ROR1 antibody Heavy Chain Variable Region CDR1:
NiP11-1 SEQ ID NO:3 Protein Artificial oil anti-ROR1 antibody Heavy Chain Variable Region CDR2:
I II,:
SEQ ID NO:4 Protein Artificial oil anti-ROR1 antibody Heavy Chain Variable Region CDR3:
DSSSWYSGWYFDL
SEQ ID NO:5 Protein Artificial oil anti-ROR1 antibody ROR1 antibody Light Chain Variable Region AIQMTQSPS SL SASVGDRVT I TCRASQGIRTDLAWYQQKPGKAPKL LIYAAS SLQSGVPSRFSGSGSG
TDFTLT IS SLQPEDFATYYCQQYYGYP IAFGQGTRLE IK
SEQ ID NO:6 Protein Artificial oil anti-ROR1 antibody Light Chain Variable Region CDR1 RAS Q. GIRT D.LA
SEQ ID NO:7 Protein Artificial oil anti-ROR1 antibody Light Chain Variable Region CDR2 ASSLCS
SEQ ID NO:8 Protein Artificial oil anti-ROR1 antibody Light Chain Variable Region CDR3 QQYYGYP IA
SEQ ID NO:9 Protein Artificial oil anti-ROR1 single chain antibody (scFv) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI INPTSGRTSYAQKFQGRV
TMTRDT ST S TVYMELS SLRSEDTAVYYCARDS SSWYSGWYFDLWGQGTTVTVS S GGGGSGGGGSGGGG
SGGGGSAIQMTQSPSSLSASVGDRVT I TCRASQGI RTDLAWYQQKP GKAPKLL IYAAS SLQSGVPSRF
SGSGSGTDFTLT S SLQPEDFATYYCQQYYGYP IAFGQGTRLE K
SEQ ID NO:10 Protein Artificial slO anti-ROR1 antibody Heavy Chain Variable Region QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRV
TMTRDT ST S TVYMELS SLRSEDTAVYYCARSSRS SYYLWVLDLWGQGTTVTVS S
SEQ ID NO: II
Protein Artificial slO anti-ROR1 antibody Heavy Chain Variable Region CDR1 SEQ ID NO:12 Protein Artificial slO anti-ROR1 antibody Heavy Chain Variable Region CDR2 I P (.,_= 'GS '1' K
SEQ ID NO:13 Protein Artificial slO anti-ROR1 antibody Heavy Chain Variable Region CDR3 SSRSSYYLWVLDL
SEQ ID NO:14 Protein Artificial slO anti-ROR1 antibody Light chain Variable Region AIQMTQSPSSLSASVGDRVTITCRASQGVSTEIAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG
TDFTLT I S SLQPEDFATYYCQQFNSYP I T FGQGTRLE I K
SEQ ID NO:15 Protein Artificial slO anti-ROR1 antibody Light chain Variable Region CDR1 RAS QGVS T E IA
SEQ ID NO:16 Protein Artificial slO anti-ROR1 antibody Light chain Variable Region CDR2 AASSLQS.
SEQ ID NO:17 Protein Artificial slO anti-ROR1 antibody Light chain Variable Region CDR3 QQFNSYPIT
SEQ ID NO:1 8 Protein Artificial slO anti-ROR1 single chain antibody (scFy) QVQLVQSGAEVKKPGASVKVSCKAS GYT FTNYYMHWVRQAPGQGLEWMGI INPSGGSTSYAQKFQGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARSSRSSYYLWVLDLWGQCTTVTVSSCGGGSGGGCSGGCG
SGGGGSAIQLTQSPSSLSASVGDRVT I TCRASQGVSTE IAWYQQKPGKAPKLL IYAAS SLQSGVPS RF
SGSGSGTDFTLT I S SLQPEDFATYYCQQFNSYP I TFGQGTRLE IK
SEQ ID NO:19 Protein Artificial j1v1011 anti-ROR1 antibody Heavy chain Variable region:
QVQLVQSGAEVKKPGASVKVSCKAS GYT FT SKYYHWVRQAPGQGLEWMGI INPTSGSTSYAQKFQGRV
TMTRDTSTSTVYMELS SLRSEDTAVYYCARDS SRYSGWYFDLWGQGTTVTVS S
SEQ ID NO:20 Protein Artificial j1v1011 anti-ROR1 antibody Heavy chain Variable region CDR1:
SKYYI-i SEQ ID NO:21 Protein Artificial j1v1011 anti-ROR1 antibody Heavy chain Variable region CDR2:
IN TS GS S ..rAQ '<EQ. G

SEQ ID NO:22 Protein Artificial j1v1011 anti-RORI antibody Heavy chain Variable region CDR3:
DSSRYSGWYFDL
SEQ ID NO:23 Protein Artificial j1v1011 anti-ROR1 antibody Light chain Variable region:
AIQLTQSPS SL SASVGDRVT I TCRASQGVSTE IAWYQQKPGKAPKL LIYAAS SLQSGVPSRFSGSGSG
TDFTLT IS SLQPEDFATYYCQQYYGYP IAFGQGTRLE IK
SEQ ID NO:24 Protein Artificial j1v1011 anti-ROR1 antibody Light chain Variable region CDR1 RA SQGVST .E a A
SEQ ID NO:25 Protein Artificial j1v1011 anti-ROR1 antibody Light chain Variable region CDR2 24ASS.LQS
SEQ ID NO:26 Protein Artificial jlvl 011 anti-ROR1 antibody Light chain Variable region CDR3 QQYYGYPIA
SEQ ID NO:27 Protein Artificial j1v1011 single chain antibody (scFv) QVQLVQSGAEVKKPGASVKVSCKAS GYT FT SKYYHWVRQAPGQGLEWMGI INPTSGSTSYAQKFQGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSRYSGWYFDLWGQGTTVTVSSGGGGSGGGGSGGGGS
GGGGSAIQLTQS PS SLSASVGDRVTI TCRASQGVSTE IAWYQQKPGKAPKLL IYAASS LQSGVPSRFS
GSGSGTDFTLT IS SLQPEDFATYYCQQYYGYP IAFGQGTRLE IK
SEQ ID NO:28 Protein Artificial Signal peptide MEWSWVFLFFLSVTTGVHS

SEQ ID NO:29 Protein Artificial (G4S)4 linker GGGGSGGGGSGGGGSGGGGS
SEQ ID NO:30 Protein Artificial Alternative GS Linker GGGSGGGSGGGSGGGSG
SEQ ID NO:31 Protein Artificial (G4S)1 Linker GGGGS
SEQ ID NO:32 Protein Artificial Hum291 anti-CD3 antibody Heavy chain Variable region:
QVQLVQSGAEVKKPGASVKVSCKASGYTFI SYTMHWVRQAPGQGLEWIGYINPRSGYTHYNQKLKDRA
TLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSS
SEQ ID NO:33 Protein Artificial Hum291 anti-CD3 antibody Light chain Variable region:
DIQMTQSP S SL SASVGDRVT I TCSAS S SVSYMNWYQQKPGKAPKRL IYDT SKLASGVP SRFS GSGS
GT
DFTLT I SSLQPEDFATYYCQQWSSNP PT FGGGTKVE IK
SEQ ID NO:34 Protein Artificial Hum291 anti-CD3 single chain antibody (scFv) OVOLVOSGAEVKKPGASVKVSCKAS GYT F I SYTMHWVRQAPGQGLEWIGYINPRSGYTHYNOKLKDRA
TLTADKSAS TAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGT LVTVS SGGGSGGGSGGGSGGGS
GDIQMTQS P S SL SASVGDRVT I TCSAS S SVSYMNWYQQKPGKAPKRL IYDT SKLASGVP SRF
SGSGSG
TDFTLT IS SLQPEDFATYYCQQWS SNP PT FGGGTKVE IK
SEQ ID NO:35 DNA
Artificial Encodes oil anti-ROR1/anti-CD3 Bispecific Antibody precursor having signal peptide - anti-ROR1 clone 011 scFv ¨ linker - anti-CD3 clone hum291 scFv ATGGAATGGAGT TGGGTT T TCT TGTT T T TCCT TAGT GTCACCACGGGAGTCCACAGCCAAGTACAACT
GGT GCAGAGT GGTGCAGAAGT CAAGAAACCTGGCGCTAGCGT GAAGGT CT CCT GTAAAGCAAGT GGAT
ATACGTTCACAAATTATTACATGCACTGGGTCCGCCAAGCTCCCGGTCAAGGGCTGGAATGGATGGGC
ATTATAAACCCCACGTCAGGCCGAACCTCCTATGCACAAAAATTCCAGGGTAGAGTGACCATGACCAG
GGATACGTCCACAAGTACAGT T TACAT GGAGCT T TCT TCACTCCGGTCT GAAGACACT GCT GTT TAT
T
ATT GCGCCCGCGATAGCTCAAGCT GGTACTCT GGAT GGTACT T T GACCT GT GGGGACAGGGGACCACC
GTGACAGTATCTTCAGGAGGCGGCGGTTCAGGTGGCGGTGGAAGCGGGGGAGGAGGCTCCGGAGGCGG
CGGATCCGCGAT TCAGAT GACGCAATCCCCAAGCAGCCTCAGT GCAAGT GTAGGCGACCGCGTTACCA
TCACTTGCCGAGCCAGTCAAGGAATACGAACCGACCTCGCCTGGTATCAGCAGAAACCTGGGAAGGCG
CCCAAACrt Crl'Arrl'ACGCCGCGT CCT CT CT CCAGAGCGGAGT GC C_;GAGT CGArl"I'T
CAGGAAGT GG
ATCT GGGACCGATT TCACACT TACAAT T TCAAGTCT TCAGCCCGAGGACT TCGCGACGTAT TAT T GCC

AACAATAT TAT GGCTATCCTATAGCAT T CGGACAAGGAACCAGGCT CGAGAT TAAAGGCGGGGGGGGC
TCTCAAGT T CAACT TGTT CAAT CT GGAGCAGAGGTAAAGAAGCCCGGCGCGAGC GTAAAGGT CT CAT G

TAAAGCCT CAGGTTATACAT T CAT TT CCTACACAAT GCACT GGGT C CGGCAGGCACCC GGT CAAGGT
C
TCGAAT GGATAGGATATAT CAATCCT CGCAGT GGCTATACT CACTATAACCAGAAGCT CAAGGAT C GA
GCCACGTT GACT GCAGATAAGT CT GCAAGTACCGCATATAT GGAAC TT T CCT CC CT CC GCT
CAGAGGA
CACT GCAGT GTACTACTGT GCACGGT CAGCATAT TACGAT TAT GAC GGAT T CGC CTAC T
GGGGACAAG
GTACACTGGT GACCGTAAGTAGTGGT GGCGGTAGT GGT GGT GGAAGCGGT GGGGGT TCCGGAGGCGGT
TCAGGT GACAT CCAAATGACT CAGAGCCCAAGCT CACT TT CCGCCT CAGTAGGGGAT C GCGT TACAAT

AACGTGCAGT GCCT CCTCAT CCGT GAGCTATAT GAACT GGTACCAACAGAAACC TGGTAAAGCT CC GA
AGCGCTTGATATATGACACGTCAAAGCTGGCTAGTGGAGTACCCAGTAGGTTTAGTGGGAGCGGGAGC
GGTACAGATTTCACTCTGACAATATCATCACTGCAACCTGAGGACT TT GCTACC TACTAT T GCCAGCA
ATGGAGTAGTAATCCGCCGACGTT TGGT GGGGGAACGAAGGT GGAGAT CAAA
SEQ ID NO:36 Protein Artificial oil anti-ROR1/anti-CD3 Bispecific Antibody precursor:
signal peptide - anti-ROR1 clone 011 scFv ¨ linker - anti-CD3 clone hum291 scFv MEW SWVFL FEL SVT TGVHSQVQLVQS GAEVKKP GASVKVS CKAS GY TFTNYYMHWVRQAP GQGL
EWMG
I INP TS GRT SYAQKEQGRVTMTRDTS T S TVYMEL S SLRSEDTAVYYCARDS S
SWYSGWYEDLWGQGTT
VTVS SGGGGSGGGGSGGGGSGGGGSAIQMTQS PS SL SASVGDRVT I TCRASQGI RTDLAWYQQKPGKA
PKLL I YAAS S LQS GVP S RFS GS GS GTDFTL TI SS LQPEDFATYYCQQYYGYP IAFGQGTRLE I
KGGGG
SQVQLVQSGAEVKKPGASVKVSCKASGYTF SYTMHWVRQAP GQGL EW GY INP RS GY THYNQKLKDR
ATLTADKSASTAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVS SGGGSGGGSGGGSGGG
S GD I QMTQS PS SL SASVGDRVT I TCSAS SSVSYMNWYQQKPGKAPKRL IYDT S KLASGVP S RFS
GS GS
GTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK
SEQ ID NO:37 DNA
Artificial Encodes slO anti-ROR1/anti-CD3 Bispecific Antibody precursor having signal peptide - anti-ROR1 clone s10 scFv ¨ linker - anti-CD3 clone hum291 scFv ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGAGCGTGACCACAGGCGTGCACTCTCAGGTTCAGCT
GGT T CAGT CT GGCGCCGAAGT GAAGAAACCTGGCGCCT CT CT GAAGGT CT CCT
GCAAGGCCAGCGGCT

ACACCTTTACCAACTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGC
ATCATCAACCCTAGCGGCGGCAGCACAAGCTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAG
AGACACCAGCACCTCCACCGTGTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACT
ACTGCGCCAGAAGCAGCAGATCCAGCTACTACCTGTGGGTGCTCGATCTGTGGGGCCAGGGAACAACC
GTGACAGTCTCTTCTGGTGGCGGAGGATCTGGCGGAGGTGGAAGCGGCGGAGGCGGTAGCGGAGGTGG
TGGATCTGCAATTCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACAA
TCACCTGTAGAGCCAGCCAGGGCGTGTCCACAGAGATCGCTTGGTATCAGCAGAAGCCCGGCAAGGCC
CCTAAGCT GCT GAT CTAT GCT GCCTCCAGT CT GCAGAGCGGCGT CC CAT CTAGATT T T CT
GGCAGC CC
CTCCGGCACCGACTTCACCCTGACAATATCTAGCCTGCAGCCAGAGGACTTCGCCACCTACTACTGCC
AGCAGTTCAACAGCTACCCCATCACCTTCGGCCAGGGCACCAGACTGGAAATCAAAGGTGGTGGTGGC
AGCCAGGT GCAGCT CGTT CAAAGCGGAGCT GAAGT GAAAAAGCCAGGGCCCACC CT CAAACT CT CT TG

CAAAGCCTCTGGCTACACATTCATCAGCTACACCATGCATTGGGTTCGCCAGGCTCCAGGCCAGGGAC
TCGAATGGATCGGCTACATCAATCCCAGAAGCGGCTATACCCACTACAACCAGAAGCTGAAGGACCGG
GCCACACTGACCGCCGATAAGTCTGCCAGCACCGCCTATATGGAACTGTCCTCTCTGCGGAGCGAAGA
TACAGCCGTGTATTATTGTGCCCGCAGCGCCTACTACGACTACGACGGCTTTGCCTAT TGGGGACAGG
GCACCCTGGTCACCGTTTCTTCTGGCGGAGGAAGTGGCGGCGGAAGCGGTGGTGGTTCTGGCGGTGGT
AGTGGCGACATCCAGATGACCCAGTCTCCAAGCTCTCTGAGCGCCAGCGTGGGCGATAGAGTCACCAT
CACATGTAGCGCCT CCAGCAGCGT GT CCTACAT GAACT GGTAT CAACAAAAGCC TGGGAAAGCT CC CA
AGCGCCTGATCTACGACACAAGCAAACTGGCCAGCGGAGTGCCCAGCAGATTTTCCGGATCTGGCAGT
GGCACAGACTTTACACTCACCATAAGCTCACTGCAGCCCGAAGATT TT GCCACGTACTAT T GTCAGCA
ATGGTCCAGCAATCCTCCTACCTTCGGAGGCGGCACCAAGGTCGAGATCAAG
SEQ ID NO:38 Protein Artificial slO anti-ROR1/anti-CD3 Bispecific Antibody precursor signal peptide - anti-ROR1 clone slO scFv ¨ linker - anti-CD3 clone hum291 scFv MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMG
I INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELS SLRSEDTAVYYCARS S RS SYYLWVLDLWGQGTT
VTVS SGGGGSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVT I TCRASQGVSTEIAWYQQKPGKA
PKLL I YAAS S LQS GVP S RFS GS GS GTDFTL TISS LQPEDFATYYCQQFNSYP IT
FGQGTRLEIKGGGG
SQVQLVQSGAEVKKPGASVKVSCKASGYTF I SYTMHWVRQAPGQGL EW I GY INP RS GY THYNQKLKDR
ATLTADKSASTAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVS SGGGSGGGSGGGSGGG
S GD I QMTQS P S SLSASVGDRVT I TCSAS SSVSYMNWYQQKPGKAPKRL IYDT SKLASGVP S RFS
GS GS
GTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK
SEQ ID NO:39 DNA
Artificial Encodes j1v1011 anti-ROR1/anti-CD3 Bispecific Antibody precursor having signal peptide - anti-ROR1 clone j1v1011 scFv¨ linker - anti-CD3 clone hum291 scFv ATGGAATGGTCCTGGGTGT TCCTGTTCT TCCTGAGCGTGACCACAGGCGTGCACTCTCAGGT TCAGCT
GGT T CAGT CT GGCGCCGAAGT GAAGAAACCTGGCGCCT CT CT GAAGGT CT CCT
GCAAGGCCAGCGGCT
ACACCTTTACCAGCAAGTACTACCACTGGGTCCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGC
ATCATCAACCCCACCAGCGGCAGCACAAGCTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAG
AGACACCAGCACCTCCACCGTGTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACT
ACTGCGCCAGAGACAGCTCTAGATACAGCGGCTGGTACTTCGACCT GTGGGGCCAGGGAACAACCGTG

ACAGTTTCTTCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGGCGGAGGAAGCGGTGGCGGCGG
ATCTGCTAT TCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCTCT GTGGGCGACAGAGTGACAAT CA
CCT GTAGAGCCT CT CAGGGCGT GT CCACAGAGAT CGCCTGGTAT CAGCAGAAGC CT GGCAAGGCCC CT

AAGCTGCTGATCTATGCCGCTAGCTCTCTGCAGTCCGGCGTGCCATCTAGATTTTCCGGCTCTGGCAG
CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCAGAGGACTTCGCCACCTACTACTGTCAGC
AGTACTACGGCTACCCTATCGCCTTCGGCCAGGGCACCAGACTGGAAATCAAAGGTGGCGGTGGCAGC
CAGGTGCAGCT CGT TCAAAGCGGAGCT GAAGT GAAAAAGCCAGGGGCCAGCGT GAAAGT GT C TT GCAA
AGCCTCTGGCTACACATTCATCAGCTACACCATGCAT TGGGT TCGCCAGGCTCCAGGCCAGGGACT CG
AATGGATCGGCTACATCAATCCCAGAAGCGGCTATACCCACTACAACCAGAAGCTGAAGGACCGGGCC
ACACTGACCGCCGATAAGTCTGCCAGCACCGCCTATATGGAACTGTCCTCTCTGCGGAGCGAAGATAC
AGCCGTGTATTATTCTGCCCGCAGCGCCTACTACCACTACCACGGCTTTGCCTATTGGGCACAGGGCA
CCCTGGTCACCGTTTCTTCTGGCGGAGGAAGTGGCGGCGGAAGCGGTGGTGGTTCTGGCGGTGGTAGT
GGCGACAT CCAGAT GACCCAGT CT CCAAGCTCT CT GAGCGCCAGCGTGGGCGATAGAGT CAC CAT CAC
ATGTAGCGCCTCCAGCAGCGTGTCCTACATGAACTGGTATCAACAAAAGCCTGGGAAAGCTCCCAAGC
GCCTGATCTACGACACAAGCAAACTGGCCAGCGGAGTGCCCAGCAGATTTTCCGGATCTGGCAGTGGC
ACAGACTT TACACT CACCATAAGCTCACT GCAGCCCGAAGAT T T T GCCACGTAC TAT T GT CAGCAAT
G
GTCCAGCAATCCTCCTACCTTCGGAGGCGGCACCAAGGTCGAGATCAAG
SEQ ID NO:40 Protein Artificial j1v1011 anti-ROR1ianti-CD3 Bispecific Antibody precursor:
signal peptide - anti-ROR1 clone j1v1011 scFv ¨ linker - anti-CD3 clone hum291 scFv MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSKYYHWVRQAPGQGLEWMG
I INP TS GS T SYAQKFQGRVTMTRDTS T S TVYMEL S SLRSEDTAVYYCARD S SRY SGWY
FDLWGQGT TV
TVS S GGGGS GGGGS GGGGS GGGGSAI QL TQ SP SSL SASVGDRVT T CRASQGVS
TEIAWYQQKPGKAP
KLL I YAAS SLQS GVPSRFS GSGSGTDFTL T I S SLQPEDFATYYCQQYYGY P IAFGQGT RLE I
KGGGGS
QVQLVQSGAEVKKPGASVKVSCKAS GYT F I SYTMHWVRQAPGQGLEWIGYINPRSGYTHYNQKLKDRA
TLTADKSAS TAYMELS SLRSEDTAVYYCARSAYYDYDGFAYWGQGT LVTVS S GGGS GGGS GGGS GGGS
GDIQMTQSPSSLSASVGDRVT I TCSAS S SVSYMNWYQQKPGKAPKRL I YDT SKLAS GVP SRF SGS GS
G

SEQ ID NO:41 DNA
Artificial EF1 ct/HTLV promoter AAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT
TGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGT
GAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTC
CTT CACGCGCCCGCCGCCCTACCT GAGGCCGCCAT CCACGCCGGT T TACT CGCGTT CT GCCGCCTC CC
GCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGT T TAAAGCTCAGGTCGAGACCGGGCCT
TTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTG
CTCAACTCTACGTCTT TGT T T CGT TT T CT GTT CT GCGCCGT TACAGAT C
SEQ ID NO:42 DNA

Cytomegalovirus CMV promoter AAGCTT GGGAGT TCCGCGT TACATAACT TACGGTAAAT GGCCCGCC TGGCT GACCGCCCAACGACCCC
CGCCCATT GACGTCAATAAT GACGTAT GT TCCCATAC TAACGCCAATACGGACT TTCCAT T GACC T CA

ATGGGT GGAGTATT TACGGTAAACTGCCCACT T GGCAGTACAT CAAGT GTAT CATAT GCCAAGTAC GC
CCCCTATT GACGTCAATGACGGTAAAT GGCCCGCCT GGCAT TAT GCCCAGTACATGACCT TATGGGAC
TTTCCTACT T GGCAGTACATCTACGTAT TAGTCATCGCTAT TACCATGGT GAT GCGGT TTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG
GGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT
CGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTACTAGA
SEQ ID NO:43 DNA
Artificial Encodes Respiratory Syncytial Virus protein F (RSV) scFv Antibody precursor:
signal peptide, VH chain ¨ linker ¨ VL chain ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGAGCGTGACCACAGGCGTGCACAGCCAAGTGACACT
GAGAGAGTCTGGCCCCGCTCTGGTCAAGCCTACACAGACCCTGACACTGACCTGCACCTTCAGCGGCT
TTAGCCTGAGCACAAGCGGCATGAGCGTCGGCTGGATTAGACAGCCTCCTGGCAAAGCCCTGGAATGG
CTGGCCGACAT T TGGT GGGACGACAAGAAGGACTACAACCCCAGCC TGAAGT CC CGGC T GAC CAT CAG

CAAGGACACCAGCAAGAACCAGGT GGT GCT GAAAGT GACCAACAT GGACCCT GC CGACACCGCCAC CT
ACTACT GT GCCAGATCCAT GAT CACCAACT GGTACT T CGACGT GT GGGGAGCCGGCAC CACAGT
GACA
GTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCTGATATCCAGATGAC
ACAGAGCCCCAGCACACT GT CT GCCAGCGT GGGAGACAGAGT GACCAT CACAT GCAAGT GCCAGCT GA
GCGT GGGCTACATGCACT GGTATCAGCAGAAGCCT GGCAAGGCCCC TAAGCT GC TGAT CTACGACACA
AGCAAGCT GGCCTCTGGCGT GCCCAGCAGATT T TCT GGCAGCGGCAGCGGAACCGAGT TCACCCTGAC
CAT CTCAAGCCT GCAGCCT GACGACT T CGCTACGTACTACT GCT TC CAAGGCAGCGGC TACCCCT T
CA
CAT T TGGAGGCGGCACCAAGCT GGAAAT CAAG
SEQ ID NO:44 Protein Artificial Respiratory Syncytial Virus protein F (RSV) scFv antibody precursor (signal peptide-VH
chain¨ linker¨VL chain) MEWSWVFLFFLSVTTGVHSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPC_;KALEW
LADIWWDDKKDYNPSLKSRLT I SKDT S KNQVVLKVTNMDPADTATYYCARSMI TNWYFDVWGAGT TVT
VS S GGGGS GGGGS GGGGS DIQMTQSPS TL SASVGDRVT TCKCQL SVGYMHWYQQKP GKAPKLL IYDT

SKLASGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCFQGSGYPFT FGGGTKLEIK
SEQ ID NO:45 Protein Artificial Respiratory Syncytial Virus protein F (RSV) scFv antibody: (VH chain¨
linker¨VL chain) QVTLRE SGPALVKP TQTL TL TCTFS GFS L S TS GMSVGW IRQP PGKALEWLAD IWWDDKKDYN PS
LKS R
LT I SKDTSKNQVVLKVTNMDPADTATYYCARSMI TNWYFDVWGAGT TVTVSSGGGGSGGGGS GGGGSD
I QMTQS PS TL SASVGDRVT I TCKCQLSVGYMHWYQQKPGKAPKLL I YDT S KLAS GVP S RFS
GSGS GTE
FTLT I SSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK
SEQ ID NO:46 DNA
Artificial Encodes human IL-12 (p40-2x elastin-p35) ATGTGCCACCAGCAGCTGGTCATCAGCTGGTTTAGCCTGGTGTTTCTGGCCTCTCCACTGGTGGCCAT
CTGGGAGCTGAAGAAAGACGT a TACGTGGTGGAACTGGACTGGTAT CCCGATGCTCCT GGCGAGAT GG
TGGTGCTGACCTGCGATACCCCTGAGGAAGAT GGCAT CACCT GGAC TCT GGACCAGT C CT CT GAGGTG
CTCGGAAGCGGCAAGACCCT GACCAT CCAAGT GAAAGAGT T T GGCGACGCCGGC CAGTACAC CT GT CA

CAAAGGCGGAGAAGTGCT GAGCCACAGCCT GCT GCT GCTCCACAAGAAAGAGGACGGCAT CT GGT C CA
CCGACATCCTGAAGGACCAGAAAGAGCCTAAGAACAAGACCTTCCTGCGCTGCGAGGCCAAGAACTAC
AGCGGCAGATTCACCTGTTGGTGGCTGACCACAATCAGCACCGACCTGACCTTCTCCGTGAAGTCTAG
CAGGGGCAGCAGTGATCCTCAGGGCGT TACATGTGGCGCCGCTACACTGTCTGCCGAAAGAGTGCGGG
GCGACAACAAAGAATACGAGTACAGCGT GGAAT GCCAAGAGGACAGCGCCT GT C CAGC CGCC GAAGAG
TCTCTGCCTATCGAAGTGATGGTGGACGCCGTGCACAAGCTGAAGTACGAGAACTACACCTCCAGCTT
TTT CAT CCGGGACATCAT CAAGCCCGAT CCTCCAAAGAACCT GCAGCT CAAGCC CCT GAAGAACAGCA
GACAGGTGGAAGTGTCTTGGGAGTACCCCGACACCTGGTCTACCCCTCACtcCTACTTCAGCCTGACC
TTT T GCGT GCAAGT GCAGGGCAAGTCCAAGCGCGAGAAAAAGGACC GGGT GT T CACCGATAAGACCAG
CGCCACCGT GAT CT GCcGAAAGAACGCCAGCAT CAGCGTCAGAGCC CAGGACCGGTAC TACAGCAGCT
CTTGGAGCGAATGGGCCAGCGTGCCATGTTCTGTGCCTGGCGTTGGAGTTCCTGGCGTGGGCAGAAAT
CTGCCAGTGGCCACGCCTGATCCTGGCATGTTTCCTTGTCTGCACCACtcCCAGAACCTGCTGAGAGC
CGTGTCCAATATGCTGCAGAAGGCCCGGCAGACACTGGAATTCTACCCCTGCACCAGCGAGGAAATCG
ACCACGAGGATATCACCAAGGACAAGACCAGCACCGTGGAAGCCTGCCTGCCTCTGGAACTGACAAAG
AACGAGAGCT GCCT GAACAGCCGGGAAACCAGCT T CAT CACCAACGGCT CT T GC CT GGCCT C
CAGAAA
GACCTCCT TCATGATGGCCCTGTGCCTGAGCAGCATCTACGAGGACCTGAAGAT GTACCAGGTGGAAT
TCAAGACCAT GAACGCCAAGCT GCTGAT GGACCCCAAGAGACAGAT CT T CCT GGACCAGAACAT GC T G

GCCGTGATCGATGAGCTGATGCAGGCCCTGAACTTCAACAGCGAGACAGTGCCCCAGAAGTCCAGCCT
GGAAGAACCCGACT TCTATAAGACCAAGAT CAAGCT GT GCAT CCT GCT GCACGC CT T C
CGGATCAGAG
CCGTGACCATCGACAGAGTGATGAGCTACCTGAACGCCTCC
SEQ ID NO:47 Protein Artificial Human IL-12 (p40-2x elastin-p35) MCHQQLVI SWFS LVFLAS PLVAIWELKKDVYVVELDWY PDAPGEMVVL TCDT PE EDGI TWTL DQS S
EV
LGSGKTLT I QVKEFGDAGQYTCHKGGEVL SHS LLLLHKKEDG IWST DI LKDQKE PKNKT FLRCEAKNY
S GRFTCWWL TT I S TDL TFSVKS SRGS S DPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEE

SLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLT
FCVQVQGKS KREKKDRVFTDKT SATVI CRKNAS I SVRAQDRYYSSSWSEWASVPCSVPGVGVPGVGRN
LPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLE FY PCT S EE IDHED TKDKTSTVEACL PLEL TK
NES CLNSRET S F I TNGSCLASRKTSFMMALCLSS I YEDLKMYQVE FKTMNAKLLMDPKRQ I FLDQNML

AVI DELMQALNFNS ETVPQKS S LEEPDFYKTKI KLC ILLHAFRI RAVT IDRVMSYLNAS
SEQ ID NO:48 DNA
Artificial encodes anti-VEGFR-2 VKl38 scFv antibody precursor (signal peptide-VH chain¨
linker¨VL
chain-IgG1 Fc region) ATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGAGCGTGACCACAGGCGTGCACTCTGAAGTGCAGCT
GGTTGAGTCTGGCGCCGAAGTGAAGAAACCTGGGAGCAGGGTGAAGGTGTCGTGCAAGGCTTAGGGCG
GCACCTTTGGCTCTTATGGCGTGTCCTGGGTTCGCAGAGCACCTGGACAAGGCCTGGAATGGATGGGC
AGACTGAT CCCCAT CT TCGGCACCAGAGACTACGCCCAGAAAT T CCAGGGCAGAGT GACCCT GACAGC
CGACGAGTCTACCAACACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACT
ACTGTGCCAGAGATGGCGACTACTACGGCAGCGGCAGCTACTATGGCATGGATGTGTGGGGCCAGGGC
ACCCTGGT TACAGT TT CT T CT GGT GGCGGAGGAT CT GGCGGAGGT GGAAGCGGC GGAGGCGGAT CT
GA
AACAACACTGACACAGAGCCCCGCCACACTGAGTGTGTCTCCAGGCGAAAGGGCCACCGTGT CT TGT c GAGCCTCTCAGAGCCTGGGCAGCAACCTCGGATGGTTCCAGCAGAAACCAGGACAGGCCCCTCGGCTG
CTGATCTATGGCGCTTCTACAAGAGCCACAGGCATCCCCGCCAGAT TT TCTGGCTCTGGCAGCGGAAC
CGAGTTCACCCTGACAATCTCTAGCCTGCAGTCCGAGGACTTCGCTGTGTACTTCTGCCAGCAGTACA
AGGACTGGCCCATGAGATTCGGCCAGGGGACCAAGGTGGAAATCAAAGAGCCCAAGAGCAGGGACAAG
ACCCACACCTGTCCTCCATGTCCTGCTCCt GAACTGCTCGGCGGACCT TCCGTGTT TCTGT T CCCT CC
AAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCC
ACGAGGACCCAGAAGT GAAGT T cAAc T GGTAt GT gGACGGCGT GGAAGT GCACAACGC CAAGACCAAG

CCTAGAGAGGAACAGTACAACAGCACCTACAGAGT GGT GT CCGT GC TGACCGT GCT GCACCAGGAT TG
GCT GAACGGCAAAGAGTACAAGTGCAAGGT GT CCAACAAGGCCCT GCCT GCT CC TAT C GAGAAAAC CA

TCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTG
ACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATG
GGAGAGCAAT GGCCAGCCAGAGAACAACTACAAGACCACT CCT CCT GTGCTGGACAGCGACGGCTCAT
TCTTCCTGTACtcCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGC
GTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCCTGT CTCTGAGCCCCGGCAAG
SEQ ID NO:49 Protein Artificial anti-VEGFR-2 VKB8 scFv-Fcl antibody precursor (signal peptide-VH chain¨
linker¨VL
chain- IgG1 Fc region) MEWSWVFL FFL SVT TGVHS EVQLVQS GAEVKKPGS SVKVS CKAYGGTFGSYGVSWVRRAPGQGLEWMG

TLVTVS SGGGGS GGGGS GGGGS ET TL TQS PATL SVS PGERATVS CRASQS L GSNLGWFQQKP
GQAP RL
LIYGASTRATGI PARFSGSGSGTEFTLT S SLQSEDFAVYFCQQYNDWP ITFGQGTKLEIKEPKSSDK
THTCPPCPAPELLGGP SVFL FP PKPKDTLM I S RT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLP PS RDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CS
VMHEALHNHYTQKS LS LS PGK
SEQ ID NO:50 DNA
Artificial anti-VEGFR-2 VKB8 scFv-Fcl antibody (VH chain¨ linker¨VL chain- IgG1 Fc region) EVQLVQSGAEVKKPGS SVKVSCKAYGGT FGSYGVSWVRRAPGQGLEWMGRL I P I FGTRDYAQKFQGRV
TLTADESTNTAYMELSSLRSEDTAVYYCARDGDYYGSGSYYGMDVWGQGTLVTVSSGGGGSGGGGS GC
GGSETTLTQSPATLSVSPGERATVSCRASQSLGSNLGWFQQKPGQAPRLL I YGASTRATGI PARFS GS
GSGTEFTLTISSLQSEDFAVYFCQQYNDWP I T FGQGTKLE I KE PKS SDKTHTCP PCPAPELLGGPSVF
LFPPKPKDTLMI SRTPEVTGVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREFOYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP IEKT I SKAKGQPREPQVYTL P P SRDEL TKNQVSL TCLVKGFYP S D
I
AVEWESNGQPENNYKT TP PVLDSDGS FFLY SKL TVDKS RWQQGNVF SCSVMHEALHNHYTQKSL SLSP
GK
SEQ ID NO:51 Protein T2A self-cleaving peptide GS GE GRGS LL T CGDVE ENP GP
SEQ ID NO:52 Protein Artificial A7 anti-ROR1 antibody Heavy Chain Variable Region QVQLVE SGGGLVKPGGS LRL SCAAS GET FS DYYMTW I RQAPGKGLEWVSY I S GS
SAYSNYADSVKGRF
T I S RDNSKNTLYLQMNS LRAEDTAVYYCARDPLLYGWL TDWGQGTLVTVS S
SEQ ID NO:53 Protein Artificial A7 anti-ROR1 antibody Heavy Chain CDR1 DYYMT
SEQ ID NO:54 Protein Artificial A7 anti-ROR1 antibody Heavy Chain CDR2 Y I S GS SAYSNYADSVKG
SEQ ID NO:55 Protein Artificial A7 anti-RORI antibody Heavy Chain CDR3 DPLLYGWLTD
SEQ ID NO:56 Protein Artificial A7 anti-ROR1 antibody Light Chain Variable Region QSAL TQPASVS GS PGQS I T I SCTGTS SVSWYQQHPGKAPKLM I YEVSKRP SGVSNRFS GS KS
GNTASL
TI S GLQAEDEADYYCS SY INDAVFEGGGTKLTVL
SEQ ID NO:57 Protein Artificial A7 anti-ROR1 antibody Light Chain CDR1 TGTS S
SEQ ID NO:58 Protein Artificial A7 anti-ROR1 antibody Light Chain CDR2 EVSKRPS
SEQ ID NO:59 Protein Artificial A7 anti-ROR1 antibody Light Chain CDR3 S SY INDAVF
SEQ ID NO:60 Protein Artificial A8 anti-ROR1 antibody Heavy Chain Variable Region QVQLVE SGGGLVKPGGS LRL SCAAS GET FS DYYMTW I RQAPGKGLEWVSY I S GS
SAYSNYADSVKGRF
T I SRDNSKNTLYLQMNSLRAEDTAVYYCARDPLLYGWL TDWGQGTLVTVS S
SEQ ID NO:61 Protein Artificial A8 anti-ROR1 antibody Heavy Chain CDR1 DYYMT
SEQ ID NO:62 Protein Artificial A8 anti-ROR1 antibody Heavy Chain CDR2 Yl S GS SAYSNYADSVKG
SEQ ID NO:63 Protein Artificial anti-ROR1 antibody Heavy Chain CDR3 DPLLYGWLTD
SEQ ID NO:64 Protein Artificial A8 anti-ROR1 antibody Light Chain Variable Region QSAL TQPASVS GS PGQS I T I SCTGTS SDGGGYDSVSWYQQHPGKAP KLMI YDVNKRP S GVS GRES
GSK

SEQ ID NO:65 Protein Artificial anti-ROR1 antibody Light Chain CDR1 TGTSSDGGGYDSVS
SEQ ID NO:66 Protein Artificial A8 anti-ROR1 antibody Light Chain CDR2 DVNKRPS

SEQ ID NO:67 Protein Artificial A8 anti-ROR1 antibody Light Chain CDR3 SSFTSDVMV
SEQ ID NO:68 Protein Artificial 2x elastin linker VPGVGVPGVG
SEQ ID NO:69 Protein Artificial (GGGGS)3 linker GGGGS GGGGS GGGGS

Claims

We claim:

1. A bispecific antibody comprising a single chain variable fragment antibody (ScFv) that binds ROR1 and a single chain variable fragment antibody (ScFv) that binds CD3, wherein the anti-RORI scFv and the anti-CD3 scFv are joined via a linker, and further wherein:
the ScFv anti-ROR1 antibody has a heavy chain variable domain having at least 95%
identity to SEQ ID NO:1 and a light chain variable domain having at least 95%
identity to SEQ ID NO:5;
the anti-ROR1 ScFv has a heavy chain variable domain having at least 95%
identity to SEQ ID NO:10 and a light chain variable domain having at least 95% identity to SEQ ID
NO:14;
the anti-ROR1 ScFv has a heavy chain variable domain having at least 95%
identity to SEQ ID NO:19 and a light chain variable domain having at least 95% identity to SEQ ID
NO:23;
the anti-ROR1 ScFv has a heavy chain variable domain having at least 95%
identity to SEQ ID NO:52 and a light chain variable domain having at least 95% identity to SEQ ID
NO:56;or the anti-ROR1 ScFv has a heavy chain variable domain having at least 95%
identity to SEQ ID NO:60 and a light chain variable domain having at least 95% identity to SEQ ID
NO:64.

2. A bispecific antibody according to claim 1, wherein the anti-ROR1 scFv has a heavy chain variable domain having at least 95% identity to SEQ ID NO:1 and a light chain variable domain having at least 95% identity to SEQ ID NO:5.

3. A bispecific antibody according to claim 2, wherein the anti-ROR1 scFv comprises an amino acid sequence having at least 95% identity to SEQ ID NO:9.

4. A bispecific antibody according to claim 1, wherein the anti-ROR1 ScFv has a heavy chain variable domain having at least 95% identity to SEQ ID NO:10 and a light chain variable domain having at least 95% identity to SEQ ID NO:14.

5. A 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.

6. A bispecific antibody according to claim 1, wherein the anti-ROR1 ScFv has a heavy chain variable domain having at least 95% identity to SEQ ID NO:19 and a light chain variable domain having at least 95% identity to SEQ ID NO:23.

7. A bispecific antibody according to claim 6, wherein the anti-ROR1 scFv comprises an amino acid sequence having at least 95% identity to SEQ ID NO:27.

8. A bispecific antibody according to claim 1, wherein the anti-CD3 scFv comprises a heavy chain variable domain having at least 95% identity to SEQ ID NO:32 and a light chain variable domain having at least 95% identity to SEQ ID NO:33.

9. A 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.

10. A bispecific antibody according to any of the previous claims wherein the heavy and light chain variable domains of the scFv antibodies are joined by a GS linker.

11. A nucleic acid construct encoding an anti-ROR1/anti-CD3 bispecific antibody according to any of claims 1-10.

12. A nucleic acid construct according to claim 11, wherein the anti-ROR1/anti-bispecific antibody-encoding sequence includes a sequence encoding a signal peptide at its N-terminus.

13. A nucleic acid construct according to claim 11, wherein the anti-ROR1/anti-bispecific antibody-encoding sequence is operably linked to a promoter.

14. A nucleic acid construct according to claim 13, wherein the promoter is an EF1a promoter, a CMV promoter, a JET promoter, an RSV promoter, an 5V40 promoter, a CAG promoter, a beta-actin promoter, an HTLV promoter, or an EFla/HTLV hybrid promoter.

15. A nucleic acid construct according to claim 13, further including a polyadenylation sequence linked to the 3' end of the anti-ROR1/anti-CD3 bispecific antibody-encoding sequence.

16. A recombinant viral genome comprising a nucleic acid construct according to any of claims 11-15.

17. A recombinant oncolytic virus comprising a nucleic acid construct according to any of claims 11-15.

18. A recombinant oncolytic virus according to claim 17, wherein the oncolytic virus is a herpes simplex virus (HSV).

19. A recombinant oncolytic virus according to claim 18, wherein the oncolytic virus is an HSV-1.

20. A recombinant oncolytic HSV according to claim 19, wherein the oncolytic HSV further comprises a nucleic acid sequence encoding IL-12.

21. A recombinant oncolytic HSV according to claim 20, wherein the IL-12 is human IL-12.

22. A recombinant oncolytic HSV according to claim 20, wherein the IL-12 comprises the amino acid sequence of SEQ ID NO:47.

23. A recombinant oncolytic HSV according to claim 19, wherein the oncolytic HSV further comprises a nucleic acid sequence encoding an anti-VEGFR antibody.

24. A recombinant oncolytic HSV according to claim 23, wherein the anti-VEGFR
antibody comprises SEQ ID NO:49.

25. A 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.

26. A recombinant oncolytic HSV according to claim 25, wherein the oncolytic HSV is derived from HSV strain 17.

27. A recombinant oncolytic HSV according to any of claims 19-26, wherein the oncolytic HSV does not encode a functional ICP34.5-encoding gene.

28. A recombinant oncolytic HSV according to claim 27, wherein all or a portion of the ICP34.5-encoding gene is deleted.

29. A 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 gene locus.

30. A recombinant oncolytic virus for use in a method of treating cancer, wherein the method comprises administering an oncolytic virus according to any of claims 17-29 to a subject having cancer.

31. A recombinant oncolytic HSV according to claim 30, wherein the oncolytic virus is an oncolytic HSV.

32. A recombinant oncolytic HSV according to claim 31, wherein the method comprises administering the oncolytic HSV by intravenous, intracavitary, intraperitoneal, intratumoral, or peritumoral delivery.

33. A recombinant oncolytic HSV according to claim 32, wherein the delivery is via catheter, infusion, or injection.

34. A recombinant oncolytic HSV according to any of claims 30-33, wherein the method comprises administering more than one dose of the oncolytic HSV to the subject.

35. A recombinant oncolytic HSV according to any of claims 30-34, wherein the cancer is a solid tumor.

36. A recombinant oncolytic HSV according to any of claims 30-35, wherein the subject is a dog, horse, or primate.

37. A recombinant oncolytic HSV according to claim 36, wherein the subject is a human.

38. A pharmaceutical composition comprising a recombinant oncolytic HSV
according to any of claims 17-37 and a pharmaceutically acceptable excipient.

39. A pharmaceutical composition according to claim 33, wherein the oncolytic HSV is at a concentration of at least 106 per ml.

40. A pharmaceutical composition according to claim 34, wherein the oncolytic HSV is at a concentration of at least 107 per ml.

41. A method of treating cancer in a subject, comprising administering an oncolytic HSV or pharmaceutical composition according to any of claims 17-40 to a subject having cancer.

42. A method according to claim 41, wherein the subject is a dog, horse, or primate.

43. A method according to claim 42, wherein the subject is a human.

44. A method according to claim 41, comprising administering the oncolytic HSV
by intravenous, intra-arterial, intracavitary, intratumoral, or peritumoral delivery.

45. A method according to claim 44, wherein delivery is by catheter, by infusion, or by injection.

46. A method according to any of claims 32 ¨ 36, comprising administering more than one dose of the oncolytic HSV to the subject.

47. A method according to any of claims 41-46, wherein the cancer is a solid tumor.

48. A host cell infected with an oncolytic virus according to any of claims 17-29.

49. A host cell according to claim 48, wherein the host cell is a Vero cell, a HEK293 cell, or a BHK cell.

50. A method of producing a pharmaceutical virus composition comprising culturing a host cell according to claim 48 to produce a viral supernatant and isolating virus from the viral supernatant to produce a pharmaceutical virus composition.

51. A virus-free conditioned medium (VCFM) comprising a bispecific antibody according to any of claims 1-10.

52. A method of treating cancer comprising treating a subject with a pharmaceutical composition comprising an anti-ROR1/anti-CD3 bispecific antibody according to any of claims 1-10.

53. A method of treating cancer comprising treating a subject with a pharmaceutical composition comprising a VFCM according to claim 51.

54. A method of according to claim 53, wherein the subject is a nonhuman subject.