Mesothelin-CD3-epsilon bispecific antibodies

Abstract

The present invention is directed to mesothelin-CD3 epsilon chain (CD3e)- human Fc bispecific antibodies. The present invention provides a method for producing the bispecific antigen-binding molecule in cells with mRNA-lipid nanoparticle (LNP). This invention provides method of antibody production in cells through in vitro transcription of mRNA-LNP and in vivo intratumoral delivery of RNA-LNP antibody to provide high in vivo efficacy. The present invention is further directed to a method for treating mesothelin-positive cancer cells by administering the bispecific mesothelin-CD3e-human Fc antibody to tumors. Intratumoral delivery of mesothelin-CD3 human Fc bispecific mRNA-LNP antibodies is effective to kill mesothelin positive cancer.

Description

[0001] MESOTHELIN-CD3-EPSILON BISPECIFIC ANTIBODIES TECHNICAL FIELD

[0002] The present invention relates to mesothelin-CD3 epsilon chain (CD3e)-human Fc bispecific antibodies. The present invention also relates to a method for killing mesothelin-positive cancer cells by transfecting mRNA-lipid nanoparticle (LNP) encoding mesothelin-CD3e human Fc bispecific antibodies into cancer cells. Intratumoral delivery of mesothelin-CD3 human Fc bispecific mRNA-LNP antibodies is effective to kill mesothelin positive cancer.

[0003] BACKGROUND

[0004] Immunotherapy is emerging as a highly promising approach for the treatment of cancer. T cells or T lymphocytes, the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells. Using bispecific antibodies binding T cells and tumor associated antigen is the most common approach to design bispecific antibody by bringing cytotoxic T cells to kill cancer cells (1-3). Bispecific antibodies can be infused into patients by different routes such as intravenous, intraperitoneal and intratumorally. The advantage of bispecific antibodies compared with chemotherapy or monoclonal antibody is that it specifically targets antigen-positive cancer cells and simultaneously activates T cells (4).

[0005] Redirecting the activity of T cells by bispecific antibodies against tumor cells, independently of their TCR specificity, is a potent approach to treat cancer. The concept is based on recognition of a tumor cell surface antigen and simultaneous binding to the CD3 epsilon chain (CD3e) within the T-cell receptor (TCR) complex on T cells. This triggers T-cell activation, including release of cytotoxic molecules, cytokines and chemokines, and induction of T-cell proliferation (4).

[0006] RNA-LNP platform used to produce bispecific antibodies inside tumors in vivo is very useful and powerful as it allows to produce and manufacture antibodies inside organism in vivo and kill tumors by bringing T cells to the tumor site. We developed intratumoral production of bispecific antibodies using mRNA-LNP bringing T cells that killed tumors.

[0007] Mesothelin is a protein encoded by MSLN gene located on chromosome 16 pl3.3. It is highly expressed in epithelial cancers and has lower expression in normal epithelial tissues.

[0008] 1

[0009] 185237047 1Membrane-anchored mesothelin forms may play a role in cellular adhesion, mesothelin regulates cell-cell contact adhesions and tissue plasticity, and controls cell proliferation and differentiation.

[0010] Human mesothelin is a polypeptide having 630 amino acids; it is encoded by MSLN gene; Uniprot database # Q13421.

[0011] Mesothelin is a tumor antigen that is highly expressed in many cancers such as pancreatic, mesothelioma, ovarian and lung cancers. It is an attractive target because its expression in normal tissues is only detected in mesothelial cells lining pericardium, peritoneum, and pleura. There are several strategies to target mesothelin in cancer such as vaccine, monoclonal antibodies, immunotoxins, antibody drug candidates and CAR-T cells.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the structure of mesothelin ScFv-linker-CD3 ScFv-mutant human Fc. The antibody has one mesothelin binding moiety and one CD3 binding moiety with mutant human Fc structure having L234AL235A and P329G mutations.

[0013] FIG. 2 shows the scheme of DNA vector template (top) used for in vitro transcription to RNA (bottom). 5’UTR: 5’ untranslated region; 3’UTR: 3 ’untranslated region; poly A tail for increased stability.

[0014] FIG. 3 shows mesothelin-CD3 mRNA-LNP transfected 293 cells generate antibody that bound A1847 mesothelin-positive cells and CD3-positive T cells. FACS with supernatant from 293 cells transfected with mesothelin-CD3 hFc mRNA-LNP was used as a primary antibody, and then anti-human Fc was used as a secondary antibody.

[0015] FIGs. 4A and 4B show that transfection of mesothelin-CD3 hFc mRNA-LNP into SKOV-3 and A1847 cancer cell lines generates functional antibody that binds to mesothelin-positive A1847 (4A) and CD3-positive T cells (4B). FACS was performed with supernatant on untransfected SKOV-3 and A1847 cancer cells and cancer cells transfected with mesothelin-CD3 hFc mRNA-LNP cells. The human Fc antibody was used as a secondary antibody. Mesothelin-positive target cell line was A1847 cell line and CD3-positive cells were primary T cells for binding in FACS.

[0016] FIG. 5 shows transfection of mesothelin-CD3 hFc mRNA-LNP into cancer cell lines with T cells improved the killing of mesothelin-positive Al 847 cells (upper panel), but did not improve the killing of mesothelin-negative C30 cells (lower panel). RTCA assay was performed either with T cells alone, antibody alone, or T cells plus different dilutions of antibody supernatants.

[0017] 2

[0018] 185237047 1FIG. 6 shows mesothelin-CD3 antibody together with T cells secreted IFN-gamma with mesothelin-positive Al 847 cells but not with mesothelin-negative C30 cells. T cells with mesothelin-CD3-hFc bispecific antibody secreted IFN-gamma in a dose-dependent manner against mesothelin-positive target cells. T cells alone did not secrete IFN-gamma against mesothelin positive cells.

[0019] FIG.7 shows mesothelin-CD3-hFc mRNA-LNP injected with T cells into mice intratumorally significantly decreased mouse xenograft rumor growth in vivo. EGFR mRNA encoded enhanced green fluorescent protein (EGFP) was used as a negative control. p<0.05 mesothelin-CD3 hFc mRNA-LNP vs EGFP mRNA-LNP.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Definitions

[0022] As used herein, “affinity” is the strength of binding of a single molecule to its ligand. Affinity is typically measured and reported by the equilibrium dissociation constant (KD or Kd), which is used to evaluate and rank order strengths of bimolecular interactions.

[0023] As used herein, “bispecific antibody” is an artificial protein that can simultaneously bind to two different types of antigen or different epitopes of the same antigen.

[0024] As used herein, “CD3 epsilon (CD3e)” is a polypeptide encoded by the CD3E gene which resides on chromosome 11. CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha / beta forms the T cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 epsilon polypeptide plays an essential role in T-cell development. CD3 epsilon, CD3e, and CD3 are used interchangeably in this application.

[0025] As used herein, a "domain" means one region in a polypeptide which is folded into a particular structure independently of other regions.

[0026] As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for preparing a scFv are known to a person skilled in the art.

[0027] As used herein, a "tumor antigen" means a biological molecule having antigenicity, expression of which causes cancer.

[0028] 3

[0029] 185237047 1Bispecific antigen-binding molecule

[0030] The present invention is directed to bispecific antibodies that specifically binds to both human mesothelin and human CD3e. The mesothelin-CD3e human Fc bispecific antibody targets mesothelin tumor antigen which is highly overexpressed in many types of cancer such as ovarian, pancreatic, and colon cancer. The mesothelin-CD3 bispecific antibodies of the present invention have high cytotoxic activity against mesothelin-positive ovarian cancer cell line and does not have activity against mesothelin-negative ovarian cancer cell line. The bispecific antibody activates T cells and re-directs T cells to mesothelin-positive cancer cells to kill cancer cells.

[0031] FIG. 1 shows a bispecific antibody structure of monovalent humanized mesothelin ScFv and monovalent CD3e ScFv fused to a human Fc domain; the structure consists of one DNA construct. The structure has a mesothelin scFv and CD3e scFv connected by a linker, and then a Fc domain (a hinge linker-CH2-CH3) fused to CD3 scFv to increase stability. The bispecific antibody (BITE-human Fc format) comprises one binding moiety to mesothelin, and one binding moiety to CD3 epsilon. The antibody may have a dimeric conformation.

[0032] The present invention provides a bispecific antigen-binding molecule comprising mesothelin VH, a first linker, mesothelin VL, a second linker, CD3 VH, a third linker, CD3 VL, and a Fc domain.

[0033] In one embodiment, the mesothelin VH has the amino acid sequence of SEQ ID NO: 8, the mesothelin VL has the amino acid sequence of SEQ ID NO: 14, the CD3 VH has the amino acid sequence of SEQ ID NO: 18, and the CD3 VL has the amino acid sequence of SEQ ID NO: 22.

[0034] In one embodiment, the Fc domain comprises a hinge linker, and CH2-CH3 of human IgGl, optionally substituted with one or two amino acid substitutions.

[0035] In one embodiment, the Fc domain comprises one or more amino acid substitutions selected from the group of L234A, L235A, and P329G (EU numbering); the amino acid substitution is to improve stability and to reduce Fc-dependent ADCC immune response.

[0036] The linkers of the present bispecific antigen-binding molecules can have the same or different amino acid sequences. In one embodiment, the linker has the amino acid sequence of (GGGGS)n, SEQ ID NO: 25, and n= 1-5. In one preferred embodiment, n= 3.

[0037] In one embodiment, the bispecific antigen-binding molecule has the amino acid sequence of SEQ ID NO: 2.

[0038] This invention generated mesothelin-CD3e bispecific antibody binding mesothelin and T cells to kill tumor cells. The delivery is done inside tumors using RNA-LNP that has

[0039] 4

[0040] 185237047 1the advantage of economical manufacturing antibody inside organism, local delivery to tumor and high efficacy.

[0041] Method for preparing bispecific antibody using mRNA-LNP.

[0042] The nucleic acid encoding an antibody or an antigen-binding fragment thereof can be inserted into a vector and expressed in mammalian 293S or CHO cells using serum-free medium. The antibody with human Fc can be purified with protein A or protein G column and used for the study. The antibody with His tag can be purified with single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used.

[0043] The present invention provides an isolated DNA sequence comprising (a) a promoter coding sequence, (b) 5'-UTR (untranslated region) coding sequence, (c) a coding sequence to encode an antibody or an antigen-binding fragment thereof, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence. In one embodiment, the antibody or the antigen-binding fragment thereof is the bispecific antigen-binding molecule of the present invention.

[0044] In the DNA sequence, the promoter is T7, T7AG promoter or SP6 promoter. Poly A tail sequence is from 20-170 nucleotides. Poly A tail sequence optionally comprises one or more linkers in between the poly A segments. If poly A tail is longer than 60 nucleotides, then it typically contains a linker which includes non-adenosine nucleotides. A linker is 5-30 or 5-25 nucleotides, e.g., 10 nucleotides or 20 nucleotides. In one example, poly A tails is 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence, and another 70 adenosine residues. In another example, poly A tails is 90 nucleotides in length, consisting of a stretch of 40 adenosine residues, followed by a 30-nucleotide linker sequence, and another 30 adenosine residues. In yet another example, poly A tail is 150-160 nucleotides in length, consisting of a two linker sequences.

[0045] DNA expression is finely regulated at the post-transcriptional level. Untranslated regions are not translated into amino acids; however, UTRs of mRNAs may control their translation, degradation and localization include stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites and various cis-acting elements that are bound by RNA-binding proteins. UTRs are important in the post-transcriptional regulation of DNA expression, including modulation of the transport of mRNAs out of the nucleus and of translation efficiency, subcellular localization, and stability.

[0046] 5

[0047] 185237047 15’-UTR typically has 10-1000 nucleotides, or 20-500 nucleotides, or 30-200 nucleotides, or 30-100 nucleotides. For example, 5’-UTR is 50 nucleotides. 3’-UTR typically has 10-3000 nucleotides, for example, 50-500 nucleotides, or 100-300 nucleotides. Preferred 5’-UTRs and 3’-UTRs are UTRs of 0-globin, or UTRs of Pfizer CO VID vaccine.

[0048] P-Globin gene is shown in:

[0049] https: / / www.ncbi.nhn.nih. £ov / nucleotide / V00497.1?report=genbank&log$=nuclalign&blast

[0050]

[0051] rank=5& RID=TDDZlK98016 In one embodiment, the 5 '-untranslated region is derived from human alpha-globin RNA with an optimized Kozak sequence. The 3' untranslated region comprises two sequence elements derived from the amino-terminal enhancer of split (AES) mRNA and the mitochondrial encoded 12S ribosomal RNA to confer RNA stability and high total protein expression.

[0052] Any suitable vector, such as Vector pSP64 Poly(A) (Promega) or pGEM3Z-Vector (Promega) can be used as a cloning vector for the DNA sequence described above.

[0053] For example, to engineer the pEM3Z-0-globin UTR-UTR-poly A tail, the 3’-UTR of the p)-globin molecule flanked by restriction enzyme site can be amplified from human bone marrow. For example, a single (pEM3Z-i -globin-UTR-A

[0120] ) or 2 serial fragments (pEM3Z-2P-globin-UTR-A

[0120] ) can be inserted in front of the poly(A) tail.

[0054] The present invention provides a method for producing an antibody or an antigenbinding fragment thereof in cells and, also in vivo inside the body. The method comprises the steps of: obtaining the DNA sequence as described above, transcribing the DNA sequence to mRNA with RNA polymerase in vitro, electroporating or embedding RNA into LNP and transfecting the mRNA into cells or into tumors, and translating the mRNA in the cells to produce the antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or the antigen-binding fragment thereof is the bispecific antigen-binding molecule of the present invention.

[0055] FIG. 2 shows the scheme of DNA vector template (top) used for in vitro transcription to RNA (bottom).

[0056] mRNA can be embedded to LNP to transfect human cells.

[0057] The inventors demonstrated that mesothelin-CD3 antibody can be produced using in vitro transcription of DNA template to produce antibody using RNA-LNP transfected into 293 cells.

[0058] 6

[0059] 185237047 1Antibodies also can be produced using adenoviruses or other viruses inside the cells providing in vivo manufacturing which decreases cost of manufacturing, generating stable cell lines for production of antibodies.

[0060] In one embodiment, antibody DNAs are inserted into DNA template vector with either T7 or SP6 promoter for RNA polymerase to transcribe to antibody RNAs by in vitro transcription. RNA is mixed with lipid components to produce lipid nanoparticles (LNPs) with RNA encapsulated. Then LNP-encapsulated mRNAs are electroporated or transfected into tumor cells or mammalian cells to translate mRNAs inside cells to produce antibody protein, which can be used for killing of tumor cells.

[0061] In general, mRNA is transient and short-lived when delivered in vivo. The present invention provides a method for producing an antibody or antigen-binding molecule by delivering lipid nanoparticle-encapsulated mRNA of an antibody or an antigen-binding molecule in cells. By encapsulating mRNA in lipid nanoparticles, the stability of mRNA is improved. The method comprises the steps of: (i) obtaining a DNA sequence comprising: (a) a promoter coding sequence, (b) 5'-UTR (untranslated region) coding sequence, (c) a sequence to encode an antigen-binding molecule of the present invention, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence; (ii) transcribing the DNA sequence to mRNA with RNA polymerase in vitro, (iii) encapsulating the mRNA in lipid nanoparticles (LNPs), (iv) transfecting the mRNA-encapsulated LNPs into cells, and (v) translating the mRNA in the cells to produce the antigen-binding molecule.

[0062] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG2000). [LNP-102 (ii)]

[0063] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl] amino] -octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-MAL).

[0064] [LNP-102 (i)]

[0065] In one embodiment, the lipid nanoparticles comprise 2-hexyLdecanoic acid, 1, l'-[[(4-hydroxybutyl)imino]di-6,l -hexanediyl] ester (ALC-0315), DSPC, Cholesterol, and a-[2-(ditetradecylamino)-2-oxoethyl]-co-methoxy-poly(oxy-l,2-ethanediyl) (ALC-0159). [LNP-315]

[0066] 7

[0067] 185237047 1Insertion of mRNA into LNP nanoparticles provides protection of mRNA from degradation and increases the stability mRNA; mRNA is then released from LNPs into cells in vivo to generate protein. mRNA-lipid nanoparticle preparation is described in Schoenmaker (International J. Pharmaceutics, 601: 120856, 2021), the article is incorporated herein by reference in its entirety, in particular regarding the LNPs.

[0068] Method for treating cancer

[0069] The present invention is directed to a method for treating cancer, comprising the step of administering a bispecific mesothelin-CD3e antigen-binding molecule or antibody to a subject suffering from cancer by intratumor injection, wherein the cancer is selected from the group consisting of ovarian cancer, lung cancer, pancreatic cancer, stomach cancer, testicular cancer, teratoma, seminoma, and cervical cancer.

[0070] In one embodiment, the method comprises the steps of; obtaining an mRNA sequence comprising a coding sequence to encode a bispecific mesothelin-CD3e antigen-binding molecule; mixing the mRNA with lipid nanoparticles to form a mRNA-lipid nanoparticle complex; and injecting the mRNA-lipid nanoparticle complex into cancer cells.

[0071] In one embodiment, the bispecific mesothelin-CD3e antigen-binding molecule has a sequence as described above in this application.

[0072] In one embodiment, the method further comprises injecting T cells intravenously. In one embodiment, RNA is embedded into LNPs and transfected into normal cells, cancer cells, and inside tumors in vivo. The lipid nanoparticle-based drug delivery method demonstrates many advantages, such as high bioavailability, solubility, stability, passage through the blood-brain barrier, and low toxicity and minimal side effects.

[0073] This present method generates mesothelin-Cd3 e bispecific antibody that binds mesothelin-positive cancer cells and T cells to kill cancer cells. The delivery is done inside tumors using RNA-LNP that has the advantage of economical manufacturing antibody inside organism, local delivery to tumor and high efficacy.

[0074] The present disclosure demonstrates the efficacy of bispecific antibodies targeting mesothelin antigen overexpressed in cancer tumors. This disclosure demonstrates that mesothelin-CD3e antibody binds CD3e antigen and mesothelin antigen. This antibody delivered with T cells specifically decreases viability of mesothelin-positive ovarian cancer cells, but not mesothelin-negative ovarian cancer cells. Mesothelin-CD3e mutant Fc antibody delivered with T cells causes secretion of significant level of IFN-gamma after coincubation with mesothelin-positive ovarian cancer cells, but not after co-incubation with 8

[0075] 185237047 1mesothelin-negative target ovarian cancer cells. This disclosure demonstrates that mesothelin-CD3e human Fc antibody administered with T cells significantly decreases ovarian Al 847 (mesothelin-positive cancer cells) tumor growth in vitro.

[0076] The present disclosure demonstrates that mesothelin-mutant hFc antibody with T cells significantly kill mesothelin-positive cancer cells, but do not kill mesothelin-negative ovarian cancers. This implies high specificity of mesothelin-CD3 -mutant hFc antibody.

[0077] The present disclosure demonstrates that mesothelin-CD3-mutant hFc RNA-LNP transfected to cancer cell lines produces antibodies.

[0078] The antibody also can also be made without Fc (BITE) for shorter sequence and shorter half-life.

[0079] The present disclosure demonstrates that mesothelin-CD3 -mutant hFc antibody RNA-LNPs injected into A 1847 tumors with T cells significantly decreases xenograft tumor growth. This shows in vivo production of antibody from injected RNA-LNP and shows its high in vivo efficacy.

[0080] Different immunomodulators can be added to increase activity of T cells and other immune cells for higher efficacy of the therapy.

[0081] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as limiting.

[0082] EXAMPLES

[0083] Example 1. Materials and Methods

[0084] Cells and culture medium

[0085] HEK293 S cells from AlStem (Richmond, CA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% FBS and 1% penicillin / streptomycin. Human peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained from the Stanford Hospital Blood Center, Stanford, CA according to IRB -approved protocol using Ficoll-Paque solution (GE Healthcare). T cells were expanded from PBMC with CD3-CD28 beads in medium with IL-2 as described below (5). Ovarian cancer cell lines: mesothelin-negative: C30 and mesothelin-positive: A 1847 cells and SKOV-3 obtained from ATCC were used for the study. The cells were cultured in a humidified 5% CO2 (6).

[0086] 9

[0087] 185237047 1Antibodies

[0088] The (APC)-labeled anti-CD3 and secondary antibodies were described in (7).

[0089] PBMC PBMC were resuspended at 1 x 106cells / ml in AIM V-AlbuMAX medium (Thermo Fisher) containing 10% FBS with 300 U / ml IL-2 (Thermo Fisher). PBMC cells were activated with CD3 / CD28 Dynabeads (Invitrogen) and used for cytotoxicity analysis with bispecific antibodies.

[0090] Fluorescence-activated cell sorting (FACS) analysis

[0091] The allophycocyanin (APC)-labeled anti-CD3 (eBioscience, San Diego, CA) antibody was used for FACS analysis using FACSCalibur (BD Biosciences). For FACS with cancer cell lines to detect mesothelin levels bi-specific MESOTHELIN-CD3 antibody and Antihuman Fc antibodies were used and analyzed on FACSCalibur, as described in (6).

[0092] Real-time cytotoxicity assay (RTCA)

[0093] Adherent ovarian cancer target cells (10,000 cells per well) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA) and cultured overnight using the impedancebased real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences). After 20-24 hours, the medium was replaced with 1 x 10⁵ effector cells T cells, T cells with bispecific antibody or antibody alone in AIM V-AlbuMAX medium containing 10% FBS, in triplicate. The cells were monitored for >40 hours with the RTCA system, and impedance (proportional to cell index) was plotted over time. Cytotoxicity was calculated as (impedance of target cells without effector cells - impedance of target cells with effector cells) xlOO / impedance of target cells without effector cells.

[0094] ELISA assay for cytokine secretion

[0095] The target cells were cultured with the effector cells or agents at in U-bottom 96-well plates with AIM V-AlbuMAX medium plus 10% FBS, in triplicate. After 16 hours, the supernatant was removed and centrifuged to remove residual cells. In some experiment, supernatant after RTCA assay was used for ELISA cytokine assays. The supernatant was transferred to a new 96-well plate and analyzed by ELISA for human cytokines using kits from Thermo Fisher according to the manufacturer’s protocol. The EC50 was calculated with GraphPad Prism software.

[0096] 10

[0097] 185237047 1Example 2. Preparation of linearized DNA template for in vitro transcription.

[0098] DNA was digested with appropriate restriction Bgl II (AGATCT) or Asc I (GGCGCGCC) enzyme which cut DNA right 3’ after poly A tail at 37°C overnight following manufacturer’s protocol. Then digested DNA was treated with 50–100 µg / mL Proteinase K and 0.5% SDS for 30 minutes at 50°C. Then phenol / chloroform extraction and ethanol precipitation of DNA was performed. The DNA was used for in vitro RNA transcription reaction.

[0099] Example 3. Preparation of RNA by in vitro transcription reaction.

[0100] 3.1. The in vitro transcription reaction was done by below protocol

[0101] When DNA template for generating RNA had T7AG promoter in front of protein or antibody coding sequence, the Standard RNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080) was used and described below: 1. Set up the following reaction at room temperature in the following order:

[0102] 20 µl Final conc.or

[0103] Components

[0104] reaction amount

[0105] Nuclease-free water X µl

[0106] 10X T7 CleanCap Reagent AG

[0107] 2 pl

[0108] Reaction Buffer

[0109] ATP (60 mM) 2 µl 6 mM final

[0110] UTP (50 mM) 2 µl 5 mM final

[0111] CTP (50 mM) 2 µl 5 mM final

[0112] GTP (50 mM) 2 µl 5 mM final

[0113] Cap Analog (40 mM) 2 µl 4 mM final

[0114] Template DNA X µl 1 µg

[0115] T7 RNA Polymerase Mix 2 µl

[0116] 2. Gently mix the reaction by pipetting up and down and microfuge briefly. Incubate at 37°C for 2 hours.

[0117] 3. Bring the reaction volume up to 50 µl with nuclease-free water. Add 2 µl of DNase I, mix well and incubate at 37°C for 15 minutes.

[0118] 4. Proceed with mRNA purification.

[0119] 11

[0120] 185237047 13.2. Cleaning in vitro transcribed RNA

[0121] For cleaning RNA, we used MEGAclear™ Kit (Thermofisher AM1908) following below protocol:

[0122] 1. Bring the RNA sample to 100 µL with Elution Solution and mix.

[0123] 2. Add 350 µL of Binding Solution Concentrate to the sample and mix.

[0124] 3. Add 250 µL of 100% ethanol to the sample.

[0125] 4. Apply the sample to the filter:

[0126] a. Insert a Filter Cartridge into one of the Collection and Elution Tubes supplied. b. Pipet the RNA mixture onto the Filter Cartridge.

[0127] c. Centrifuge for ~15 sec to 1 min, or until the mixture has passed through the filter. Centrifuge at 10,000-15,000 x g (typically 10,000-14,000 rpm).

[0128] d. Discard the flow-through and reuse the Collection and Elution Tube for the washing steps.

[0129] 5. Wash with 2 × 500 µL Wash Solution.

[0130] 6. Elute RNA from the filter with 50 µL Elution Solution

[0131] a. Pre-heat 110 µL of Elution Solution per sample to 95° C

[0132] b. Apply 50 µL of the pre-heated Elution Solution to the center of the Filter Cartridge, close the cap of the tube and centrifuge for 1 min at room temperature (RCF 10,000-15,000 x g) to elute the RNA.

[0133] c. To maximize RNA recovery, repeat this elution procedure with a second preheated 50 µL aliquot of Elution Solution. Collect the eluate into the same Collection / Elution Tube.

[0134] 3.3. Assessing RNA yield

[0135] The concentration of RNA is determined by diluting an aliquot of the preparation (usually a 1:50 to 1:100 dilution) in IxTE (10 mM Tris-HCl pH 8, 1 mM EDTA) buffer, and reading the absorbance in a spectrophotometer at 260 nm. The concentration (pg / mL) of RNA is therefore calculated as follows: A260 x dilution factor x 40 pg / mL.

[0136] Example 4. Preparation of LNPs

[0137] Lipids:

[0138] SM-102: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester; CAS number: 2089251-47-6

[0139] DMG-PEG2000: 1,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 DSPC: Distearoylphosphatidylcholine

[0140] 12

[0141] 185237047 1Cholesterol

[0142] SM-102 formulation:

[0143] The SM-102 formulation was prepared using SM-102: DSPC: Cholesterol: DMG-PEG2000= (50:10:38.5:1.5 mol%). In other experiments, different ratios and different lipid components can be used to make LNPs.

[0144] Example 5. RNA encapsulation to LNPs

[0145] Encapsulation of RNA into LNPs used either Flex S or FlexM (PreciGenome) Systems.

[0146] of RNA into LNP with Flex S System:

[0147] (organic solution): 40ul of Lipid Mix

[0148] (Aqueous Solution): 160 µl of mRNA in 100mM sodium acetate (pH:4.0) (mix 32 µg of mRNA with sodium acetate solution to bring final volume to 160 µl)

[0149] Settings:

[0150] Flow rate ratio: 4:1 (Aqueous:organic)

[0151] Total flow rate: 3ml / min

[0152] - N / P ratio: 11.0

[0153] Outlet PBS buffer dilution volume equal to total input volume (200 pl for the above example)

[0154] Encapsulation of RNA into LNP FlexM System:

[0155] (organic solution): 625 pl of Lipid Mix

[0156] (Aqueous Solution): 1875 µl of mRNA in 100mM sodium acetate (pH:4.0) (mix 500 µg of mRNA with sodium acetate solution to bring final volume to 1875 µl)

[0157] Settings:

[0158] Flow rate ratio: 3:1 (Aqueous: organic)

[0159] Total flow rate: 3ml / min

[0160] N / P ratio: 11.0

[0161] 13

[0162] 185237047 1Buffer Exchange and Filtration of RNA-LNP

[0163] After encapsulation is complete, collected samples are filtered and buffer exchange into PBS is performed using Amicon® Ultra-15 Centrifugal Filter Units (30kDA-100kDA). Fill the filter unit with ~14ml PBS and directly add your sample to the filtration unit. Spin at 1000xg for 20 minutes. Remove the flow-through solution, repeat one more time. Collect nanoparticles in PBS) from the upper compartment of the filter unit.

[0164] Flex(M) and Flex(S) samples are prepared two-fold diluted in PBS from their initial formulation volume.

[0165] The Size of LNP Using Dynamice Light Scattering (DLS) System.

[0166] The size of Nanoparticles is confirmed using Dynamic Light Scattering (DLS) system. The size of RNA-LNP nanoparticles is usually in the range of 90-130 nM.

[0167] Example 6. mRNA-LNP delivery to cells

[0168] 2 µg of encapsulated mRNA was added per 1x106HEK-293 cells in a volume of 1ml. After transfection was completed, the cells were maintained in culture medium at 37°C, 5.0% CO2.

[0169] For antibody or protein production, HEK-293 cells were kept at 37°C with shaking at 200 rpm, and then super was collected at 24-72 hours with secreted antibody or immunostimulant protein for in vitro functional assay.

[0170] Example 7. The sequences of Humanized Mesothelin-CD3e human Fc bispecific antibody

[0171] FIG. 1 shows the structure of humanized mesothelin ScFv-linkerG4Sx3- CD3 Scfv-mutant human Fc; the in vitro transcription uses one DNA template construct.

[0172] PMC2086

[0173] Template for mRNA in vitro transcription includes T7(AG) promoter (underlined); 5’UTR regular font; coding sequence for mesothelin-CD3 hFc antibody in capital font, bold, starting with ATG start codon (underlined) and ends with stop codon TGA (underlined); then 3’UTR regular font; and 152 poly A tail with linker in the middle (italics)

[0174] Nucleotide sequence

[0175] 14

[0176] 185237047 1TAATACGACTCACTATAAGGAGAAAGCTTacatttgcttctgacacaactgtgttcactagcaacctcaaa cagacaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCC AGGTTCCACTGGCGCCGCTAGCCAGGTACAGCTGCAGCAGTCAGGTCCAGG ACTCGTGACGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGAC AGTGTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGA GAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAACG ACTATGCAGTATCTGTGAAAAGTCGAATGAGCATCAACCCAGACACATCCAA GAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGT GTATTACTGTGCAAGAGGAATGATGACTTACTATTACGGTATGGACGTCTGG GGCCAAGGGACCACGGTCACCGTCTCCTCAGGCATTCTAGGATCCGGTGGC GGTGGCAGCGGCGGTGGTGGTTCCGGAGGCGGCGGTTCTCAGCCTGTGCTG ACTCAGTCGTCTTCCCTCTCTGCATCTCCTGGAGCATCAGCCAGTCTCACCT GCACCTTGCGCAGTGGCATCAATGTTGGTCCCTACAGGATATACTGGTACCA GCAGAAGCCAGGGAGTCCTCCCCAGTATCTCCTGAACTACAAATCAGACTCA GATAAGCAGCAGGGCTCTGGAGTCCCCAGCCGCTTCTCTGGATCCAAAGAT GCTTCGGCCAATGCAGGGGTTTTACTCATCTCTGGGCTCCGGTCTGAGGAT GAGGCTGACTATTACTGTATGATTTGGCACAGCAGCGCTGCTGTGTTCGGA GGAGGCACCCAACTGACCGTCCTCTCCGGGGGAGGCGGGTCTGGGGGAGG CGGAAGTGGGGGAGGAGGAAGCGAAGTTCAGCTGCTCGAATCCGGCGGCG GCCTTGTTCAGCCAGGTGGTAGCTTGAGGCTCAGTTGTGCTGCATCTGGGTT TACATTCTCAACTTATGCGATGAACTGGGTGAGGCAAGCACCTGGAAAGGG ACTTGAGTGGGTCTCAAGAATTCGCTCCAAATACAACAACTATGCGACGTAT TACGCAGACTCAGTGAAAGGACGGTTTACGATATCACGGGACGATTCAAAG AATACACTGTATTTGCAGATGAATTCTCTTAGGGCCGAAGACACTGCCGTAT ACTATTGTGTACGCCACGGTAATTTTGGCAATAGCTATGTATCTTGGTTCGC GTACTGGGGCCAAGGCACCCTTGTTACTGTGTCTAGTGGGGGCGGGGGGAG TGGTGGCGGAGGAAGTGGCGGGGGGGGATCTCAAGCGGTGGTTACTCAAG AGCCCTCCCTTACTGTTTCTCCGGGCGGGACGGTCACCTTGACTTGTGGCAG TTCAACAGGGGCAGTCACTACTAGTAATTATGCGAATTGGGTCCAAGAAAAG CCGGGCCAAGCTTTCCGGGGACTCATCGGAGGAACAAATAAAAGGGCACCC GGCACACCCGCGCGCTTTTCCGGGAGTCTTCTGGGCGGCAAGGCAGCCCTC ACTCTCTCTGGGGCTCAACCTGAGGACGAGGCTGAGTACTATTGTGCCCTCT GGTACTCAAACCTGTGGGTCTTTGGAGGGGGAACCAAGCTTACGGTCTTGT CTAGAGAAAACCTGTATTTTCAGGGCACCCACACGTGCCCCCCTTGCCCAGC ACCCGAAGCCGCAGGTGGCCCATCAGTGTTTCTTTTTCCTCCAAAACCAAAA GACACACTCATGATCTCCCGGACGCCTGAGGTGACCTGTGTAGTCGTAGAC GTATCCCATGAGGACCCTGAAGTAAAGTTTAACTGGTATGTAGACGGTGTG GAAGTACACAATGCCAAGACTAAACCAAGAGAGGAACAGTATAACAGCACC TATAGGGTAGTTTCCGTGCTCACCGTTCTCCACCAAGATTGGCTTAACGGTA AAGAATATAAATGTAAGGTGTCAAATAAGGCACTCGGAGCCCCGATCGAAA AGACCATCTCTAAAGCAAAAGGACAGCCCAGGGAGCCACAAGTCTACACCC TGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGAGctcgctttcttgctgtccaatttctat taaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacattt attttcattgcagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagg gccttgagcatctggattctgcctaataaaaaacatttattttcattgcaGTCGACTCTAG A AAAAAAAAAAAAA 15

[0177] 185237047 1AAAAAAAAAAAAAAAAAAAAAAAAGGATCCCCGGGCGAGCTCCCAAAAAAAAAAAAAAA AAAAAAAAAAAAAAACCGAATTCCTGCAGCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA (SEQ ID NO: 1)

[0178] Amino acid sequence of mesothelin-CD3 mutant human Fc METDTLLLWVLLLWVPGSTGAASQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNS ATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVT PEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGIL GSGGGGSGGGGSGGGGSGGGGSQPV LTQSSSLSASPGASASLTCTLRSG1NVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQG SGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSGG GGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG EEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFG NSYVSWFAYWGOGTLVTVSSGGGGSGGGGSGGGGSOAVVTQEPSLTVSPGGTVTLTCG SSTGAVTTSNYANWVOEKPGOAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPE DEAFI CALWKSALIWFGGGT ZTVLSRENLYFOGTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

[0179] ^Signaling peptide>

[0180] Nucleotide sequence ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTT CCACTGGC (SEO ID NO: 3)

[0181] Amino acid sequence

[0182] METDTLLLWVLLLWVPGSTG (SEQ ID NO: 4)

[0183] GCCGCTAGC AAS

[0184] < Mesothelin ScFv>

[0185] Nucleotide sequence caggtacagctgcagcagtcaggtccaggactcgtgacgccctcgcagaccctctcactcacctgtgccatctccggggacagtgtct ctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagt ggtataacgactatgcagtatctgtgaaaagtcgaatgagcatcaacccagacacatccaagaaccagttctccctgcagctgaactct gtgactcccgaggacacggctgtgtattactgtgcaagaggaatgatgacttactattacggtatggacgtctggggccaagggacca cggtcaccgtctcctcaggcattctaggatccggtggcggtggcagcggcggtggtggttccggaggcggcggttctcagcctgtgc tgactcagtcgtcttccctctctgcatctcctggagcatcagccagtctcacctgcaccttgcgcagtggcatcaatgttggtccctacag gatatactggtaccagcagaagccagggagtcctccccagtatctcctgaactacaaatcagactcagataagcagcagggctctgga gtccccagccgcttctctggatccaaagatgcttcggccaatgcaggggttttactcatctctgggctccggtctgaggatgaggctgac tattactgtatgatttggcacagcagcgctgctgtgttcggaggaggcacccaactgaccgtcctc (SEQ ID NO: 5)

[0186] 16

[0187] 185237047 1Amino acid sequence QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSK WYNDYA VSVKSRMSINPDTSKNQFSLQLNSVTPEDTA VYYCARGMMTYYYGMDVWG GGTTVTVSSGILGSGGGGS'GGGGS'GGGGS'OPVLTOSSSLSASPGASASLTCTLRSGINV GPYRIYWYOOKPGSPPOYLLNYKSDSDKOQGSGVPSRFSGSKDASANAGVLLISGLR SEDEADYYCMIWHSSAAVFGGGTQLTVL (SEQ ID NO: 6)

[0188] < Mesothelin VH>

[0189] Nucleotide sequence caggtacagctgcagcagtcaggtccaggactcgtgacgccctcgcagaccctctcactcacctgtgccatctccggggacag tgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactaca ggtccaagtggtataacgactatgcagtatctgtgaaaagtcgaatgagcatcaacccagacacatccaagaaccagttctcc ctgcagctgaactctgtgactcccgaggacacggctgtgtattactgtgcaagaggaatgatgacttactattacggtatggac gtctggggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 7)

[0190] Amino acid sequence QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSK WYNDYA VSVKSRMSINPDTSKNQFSLQLNSVTPEDTA VYYCARGMMTYYYGMDVWG QGTTVTVSS (SEQ ID NO: 8)

[0191] < Mesothelin VH tail>

[0192] Nucleotide sequence

[0193] ggcattctagga (SEQ ID NO: 9)

[0194] Amino acid sequence

[0195] GILG (SEQ ID NO: 10)

[0196] < Linker, S plus G4S x3>

[0197] Nucleotide sequence

[0198] tccgggggaggcgggtctgggggaggcggaagtgggggaggaggaagc (SEQ ID NO: 11)

[0199] Amino acid sequence

[0200] SGGGGSGGGGSGGGGS (SEQ ID NO: 12)

[0201] < Mesothelin VL>

[0202] Nucleotide sequence cagcctgtgctgactcagtcgtcttccctctctgcatctcctggagcatcagccagtctcacctgcaccttgcgcagtggcatcaatgttg gtccctacaggatatactggtaccagcagaagccagggagtcctccccagtatctcctgaactacaaatcagactcagataagcagca

[0203] 17

[0204] 185237047 1gggctctggagtccccagccgcttctctggatccaaagatgcttcggccaatgcaggggttttactcatctctgggctccggtctgagg atgaggctgactattactgtatgatttggcacagcagcgctgctgtgttcggaggaggcacccaactgaccgtcctc (SEQID NO: 13)

[0205] Amino acid sequence OPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYOOKPGSPPOYLLNYKSDSDK QQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVL

[0206] S (SEQ ID NO: 14)

[0207] < Linker, S plus G4S x3>

[0208] Nucleotide sequence (SEQ ID NO: 11)

[0209] Amino acid sequence (SEQ ID NO: 12)

[0210] < CD3 ScFv>

[0211] Nucleotide sequence gaagttcagctgctcgaatccggcggcggccttgttcagccaggtggtagcttgaggctcagttgtgctgcatctgggtttacattctcaacttatgcgatgaactgggtgaggcaagcacctggaaagggacttgagtgggtctcaagaattcgctccaaatacaacaactatgcgacgtattacgcagactcagtgaaaggacggtttacgatatcacgggacgattcaaagaatacactgtatttgcagatgaattctcttagggccgaagacactgccgtatactattgtgtacgccacggtaattttggcaatagctatgtatcttggttcgcgtactggggcc aaggcacccttgttactgtgtctagtgggggcggggggagtggtggcggaggaagtggcggggggggatctcaagcggtggtta ctcaagagccctcccttactgtttctccgggcgggacggtcaccttgacttgtggcagttcaacaggggcagtcactactagtaatta tgcgaattgggtccaagaaaagccgggccaagctttccggggactcatcggaggaacaaataaaagggcacccggcacaccc gcgcgcttttccgggagtcttctgggcggcaaggcagccctcactctctctggggctcaacctgaggacgaggctgagtactattgt gccctctggtactcaaacctgtgggtctttggagggggaaccaagcttacggtcttg (SEQ ID NO: 15)

[0212] Amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYAT YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

[0213] V7V. SSGGGGSGGGGSGGGGSQ / 1 WTOEPSLTVSPGGTVTLTCGSSTGA VTTSNYANWVQ EKPGOAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAOPEDEAEYYCAEWYSNLWV FGGGTKLTVL (SEQ ID NO: 16)

[0214] < CD3 VH>

[0215] nucleotide sequence gaagttcagctgctcgaatccggcggcggccttgttcagccaggtggtagcttgaggctcagttgtgctgcatctgggtttacattctc aacttatgcgatgaactgggtgaggcaagcacctggaaagggacttgagtgggtctcaagaattcgctccaaatacaacaactat gcgacgtattacgcagactcagtgaaaggacggtttacgatatcacgggacgattcaaagaatacactgtatttgcagatgaattc tcttagggccgaagacactgccgtatactattgtgtacgccacggtaattttggcaatagctatgtatcttggttcgcgtactggggcc aaggcacccttgttactgtgtctagt (SEQIDNO: 17)

[0216] Amino acid sequence

[0217] 18

[0218] 185237047 1EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYAT YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL VTVSS (SEQ ID NO: 18)

[0219] < G4Sx3 linker>

[0220] Nucleotide sequence

[0221] ggg ggc ggg ggg agt ggt ggc gga gga agt ggc ggg ggg gga tct (SEQ ID NO: 19)

[0222] Amino acid sequence

[0223] GGGGSGGGGSGGGGS (SEQ ID NO: 20)

[0224] < CD3 VL>

[0225] Nucleotide sequence caagcggtggttactcaagagccctcccttactgtttctccgggcgggacggtcaccttgacttgtggcagttcaacaggggcagtcac tactagtaattatgcgaattgggtccaagaaaagccgggccaagctttccggggactcatcggaggaacaaataaaagggcacccgg cacacccgcgcgcttttccgggagtcttctgggcggcaaggcagccctcactctctctggggctcaacctgaggacgaggctgagta ctattgtgccctctggtactcaaacctgtgggtctttggagggggaaccaagcttacggtcttg (SEQ ID NO: 21)

[0226] Amino acid sequence

[0227] QA WTQEPSLTVSPGGTVTLTCGSSTGA VTTSNYANWVQEKPGQAFRGLIGGTNKRAPGT PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 22)

[0228] < Human Fc> containing hinge, CH2 and CH3 domains and L234A, L235A, G329G mutations; mutations are shown in bigger font and underlined

[0229] Nucleotide sequence tctagagaaaacctgtattttcagggcacccacacgtgccccccttgcccagcacccgaagccgcaggtggcccatcagtgttt ctttttcctccaaaaccaaaagacacactcatgatctcccggacgcctgaggtgacctgtgtagtcgtagacgtatcccatgaggac cctgaagtaaagtttaactggtatgtagacggtgtggaagtacacaatgccaagactaaaccaagagaggaacagtataacag cacctatagggtagtttccgtgctcaccgttctccaccaagattggcttaacggtaaagaatataaatgtaaggtgtcaaataaggc actcggagccccgatcgaaaagaccatctctaaagcaaaaggacagcccagggagccacaagtctacaccctgcccccatc ccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggga gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagct caccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgca gaagagcctctccctgtctcccgggaaatga (SEQ ID NO: 23)

[0230] Amino acid sequence SRENLYFQGTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW

[0231] 19

[0232] 185237047 1ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 24)

[0233] Example 8. DNA template for RNA to transcribe mesothelin-CD3 mutant hFc Ab The nucleotide sequence PMC2086 (see Example 7, SEQ ID NO: 1) is inserted into DNA which is template for in vitro RNA transcription.

[0234] Before in vitro transcription reaction, the sequence is linearized at the end of poly A tail. During in vitro transcription reaction, the 5’Cap is added for increased stability of RNA. The RNA is checked on agarose gel and used to transfect 293 cells to generate mesothelin-CD3-hFc antibody.

[0235] Example 9. Mesothelin Scfv-CD3 Scfv-human Fc (PMC2086) produced by RNA transcription shows good activity and specific efficacy in vitro

[0236] The mesothelin ScFV-linker-CD3 ScFv-mutant human Fc nucleotide sequence (Example 7) was used as a template for RNA transcription. The RNA was generated using In vitro transcription kit. The RNA structure generated is shown on FIG. 2. This RNA was embedded to LNP and transfected to 293 cells, and then the supernatant was collected to see on SDS gel at 72-96 hours.

[0237] The supernatant collected after 72-hour transfection of 293 cells with PMC2086 mRNA-LNP contained mesothelin-CD3-hFc antibody, which bound mesothelin-positive ovarian cancer A 1847 cells and also bound CD3-positive T cells by FACS (FIG 3.).

[0238] Example 10. Expression of mesothelin-CD3-human Fc antibody in cancer cell lines transfected with mesothelin-CD3 mRNA-LNP.

[0239] Mesothelin-CD3 mRNA-LNP was transfected to different ovarian cancer cells SKOV-3 and A1847 cell lines. We found that mesothelin-CD3 hFc was secreted from A1847 and SKOV-3 cells cancer cell line. The supernatant from SKOV-3 and A1847 cells containing this antibody bound target mesothelin-positive Al 847 cells as detected by FACS with anti-human Fc antibody (FIG. 4A). The supernatant from SKOV-3 and Al 847 cells containing this antibody also bound T cells by FACS (FIG. 4B). This demonstrates that bispecific antibodies were produced in SKOV-3 and A 1847 cells using mRNA-LNP technology and bound target mesothelin-positive cell line and T cells. The mRNA-LNP technology saves time and cost for manufacturing antibodies using cancer cells as antibody factories in vivo (inside body).

[0240] 20

[0241] 185237047 1Example 11. The mesothelin-CD3 antibody that was generated with mesothelin mRNA-LNP and transfected into A1847 cells killed mesothelin positive A1847 ovarian cancer cells but not mesothelin-negative C30 ovarian cancer cells by RTCA assay.

[0242] To test that mesothelin-CD3 hFc antibodies generated from mesothelin-CD3 RNA-LNP were functional, we used supernatant after transfection A1847 cells and used A1847, mesothelin-positive and C30 mesothelin-negative ovarian cancer, mesothelin-negative cancer cell lines in RTCA killing assay (FIG. 5). mesothelin-CD3- hFc supernatants from mRNA-LNP-transfected A 1847 cells killed mesothelin-positive target cells in a dose-dependent manner and did not kill C30 mesothelin-negative cell line (FIG. 5). No killing was observed with mesothelin-negative CHO cell line (not shown).

[0243] Example 12. T cells with mRNA-LNP encoded Mesothelin-CD3 hFc antibody secreted IFN-gamma against mesothelin-positive target cells.

[0244] The mesothelin-CD3 human Fc antibody collected from supernatant of transfected A 1847 cells were added to T cells to demonstrate secretion of IFN-gamma in a dosedependent manner by mesothelin-positive A 1847 cells, but not with mesothelin-negative C30 cells or CHO cells (FIG. 6).

[0245] Example 13. In vivo efficacy of mesothelin-CD3-human Fc-LNP with T cells in vivo in mice.

[0246] To test that mesothelin-CD3 antibodies can be generated from mesothelin-CD3 RNA-LNP in vivo, we used mesothelin-CD3-mutant hFc RNA-LNP, and injected it into mice with A1847 ovarian xenograft tumors either alone or together with T cells. We injected 2x106ovarian A1847 cancer cell line subcutaneously into NSG mice, and then injected intratumorally mesothelin-CD3-hFc-RNA-LNP on days 7, 14, and 21. Human T cells (1x107cells / mice) were delivered intravenously into mice on day 9. Mesothelin-CD3 mRNA-LNP injected into ovarian xenograft tumors with T cells significantly decreased A1847 tumor growth versus T cells alone or mesothelin-CD3 -mutant hFc RNA-LNP alone (FIG.7).

[0247] This result shows that bispecific antibodies can be generated in vivo, which significantly decreases the cost of antibody manufacturing. Bispecific mesothelin-CD3 antibodies produced after delivery of RNA-LNP and with T cells significantly decreased ovarian xenograft tumor growth.

[0248] 21

[0249] 185237047 1REFERENCES

[0250] 1. M. Bacac, T. Fauti, J. Sam, S. Colombetti, T. Weinzierl, D. Ouaret, W. Bodmer, S. Lehmann, T. Hofer, R. J. Hosse, E. Moessner, O. Ast, P. Bruenker, S. Grau-Richards, T. Schaller, A. Seidl, C. Gerdes, M. Perro, V. Nicolini, N. Steinhoff, S. Dudal, S. Neumann, T. von Hirschheydt, C. Jaeger, J. Saro, V. Karanikas, C. Klein and P. Umana: A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors. Clin Cancer Res, 22(13), 3286-97 (2016) doi:10.1158 / 1078-0432. CCR-15-1696

[0251] 2. N. Bumma, N. Papadantonakis and A. S. Advani: Structure, development, preclinical and clinical efficacy of blinatumomab in acute lymphoblastic leukemia. Future Oncol, 11(12), 1729-39 (2015) doi:10.2217 / fon.15.84

[0252] 3. S. L. Ross, M. Sherman, P. L. McElroy, J. A. Lofgren, G. Moody, P. A. Baeuerle, A. Coxon and T. Arvedson: Bispecific T cell engager (BiTE(R)) antibody constructs can mediate bystander tumor cell killing. PLoS One, 12(8), e0183390 (2017)

[0253] doi:10.1371 / journal.pone.0183390

[0254] 4. C. Klein, W. Schaefer, J. T. Regula, C. Dumontet, U. Brinkmann, M. Bacac and P. Umana: Engineering therapeutic bispecific antibodies using CrossMab technology. Methods, 154, 21-31 (2019) doi:10.1016 / j.ymeth.2018.11.008

[0255] 5. V. Golubovskaya, H. Zhou, F. Li, M. Valentine, J. Sun, R. Berahovich, S. Xu, M. Quintanilla, M. C. Ma, J. Sienkiewicz, Y. Huang and L. Wu: Novel CD37, Humanized CD37 and Bi-Specific Humanized CD37-CD19 CAR-T Cells Specifically Target Lymphoma. Cancers (Basel), 13(5) (2021) doi:10.3390 / cancersl3050981

[0256] 6. R. Berahovich, H. Zhou, S. Xu, Y. Wei, J. Guan, J. Guan, H. Harto, S. Fu, K. Yang, S. Zhu, L. Li, L. Wu and V. Golubovskaya: CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth. Cancers (Basel), 10(9) (2018) doi:10.3390 / cancersl0090323

[0257] 7. L. Wu, Y. Huang, J. Sienkiewicz, J. Sun, L. Guiang, F. Li, L. Yang and V.

[0258] Golubovskaya: Bispecific BCMA-CD3 Antibodies Block Multiple Myeloma Tumor Growth. Cancers (Basel), 14(10) (2022) doi:10.3390 / cancersl4102518

[0259] 8. L. Bergan, J. A. Gross, B. Nevin, N. Urban and N. Scholler: Development and in vitro validation of anti-mesothelin biobodies that prevent CA125 / mesothelin-dependent cell attachment. Cancer Lett, 255(2), 263-74 (2007) doi:10.1016 / j.canlet.2007.04.012

[0260] 22

[0261] 185237047 1

Claims

1. WHAT IS CLAIMED IS:

1. A bispecific antigen-binding molecule comprising mesothelin VH having SEQ ID NO: 8, a first linker, mesothelin VL having SEQ ID NO: 14, a second linker, CD3 VH having SEQ ID NO: 18, a third linker, CD3 VL having SEQ ID NO: 22, and a human Fc domain.

2. The bispecific antigen-binding molecule of claim 1, wherein the human Fc domain comprises a hinge linker, and CH2-CH3 of human IgGl, optionally substituted with one or two amino acid substitutions.

3. The bispecific antigen-binding molecule of claim 2, wherein the Fc domain comprises one or more amino acid substitutions selected from the group of L234A, L235A, and P329G (EU numbering).

4. The bispecific antigen-binding molecule of any one of claims 1-3, wherein each first, second, and third link comprise the amino acid sequence of (GGGGS)n, and n= 1-5.

5. The bispecific antigen-binding molecule of claim 1, having the amino acid sequence of SEQ ID NO: 2.

6. The bispecific antigen-binding molecule of claim 1, wherein the human Fc domain comprises the amino acid sequence of SEQ ID NO: 24.

7. An isolated DNA sequence comprising (a) a promoter coding sequence, (b) 5'-UTR (untranslated region) coding sequence, (c) a coding sequence to encode the bispecific antigen-binding molecule of any one of claims 1-6, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence.

8. The isolated DNA sequence of claim 7, having the nucleotide sequence of SEQ ID NO: 1.

9. Lipid nanoparticles (LNPs) have mRNA encapsulated, wherein the mRNA is transcribed from the isolated DNA sequence of claim 8.

10. A method for producing a bispecific antigen-binding molecule in cells, comprising the 23185237047 1steps of:obtaining the DNA sequence of claim 7 or 8,transcribing the DNA sequence to mRNA with RNA polymerase in vitro, mixing the mRNA with lipid nanoparticles (LNP) to form mRNA-LNP complex, transfecting the mRNA-encapsulated LNPs into cells, andtranslating the mRNA in the cells to produce the bispecific antigen-binding molecule in cells.

11. The method of claim 10, wherein the cells are cancer cells.

12. A method for treating cancer, comprising the steps of:obtaining the DNA sequence of claim 7 or 8,transcribing the DNA sequence to mRNA with RNA polymerase in vitro, mixing the mRNA with lipid nanoparticles (LNP) to form mRNA-LNP complex, and injecting the mRNA-lipid nanoparticle complex into tumors.

13. The method of claim 12, further comprising injecting T cells intravenously.

14. The method of claims 12, wherein the cancer is ovarian cancer, lung cancer, pancreatic cancer, stomach cancer, testicular cancer, teratoma, seminoma, or cervical cancer.24185237047 1

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