RECOMBINANT BIFUNCTIONAL PROTEIN TARGETING CD47 AND HER2
20230167194 · 2023-06-01
Inventors
Cpc classification
C07K2319/75
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K14/705
CHEMISTRY; METALLURGY
Abstract
The present disclosure provides a recombinant fusion protein containing an extracellular Ig-like domain of a signal-regulator protein (SIRP), linked via a linker, to a paratope of an Ig-like anti-HER2 antibody at the N-terminus of a heavy chain or a light chain constituting the paratope. The present disclosure also provides a polynucleotide encoding the recombinant fusion protein, an expression vector containing the polynucleotide, a method for producing the recombinant protein and a method for treating a disease caused by over-expression of CD47 and/or HER2.
Claims
1. A method for treating gastric cancer caused by over-expression of CD47 and/or HER2, comprising administering to a patient or a subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant fusion protein and at least one pharmaceutical carrier; wherein the recombinant fusion protein comprises an extracellular Ig-like domain of a signal-regulatory protein (SIRP), linked via a linker, to a paratope of an Ig-like anti-HER2 antibody at the N-terminus of a heavy chain or a light chain constituting the paratope; wherein the recombinant fusion protein is capable of blocking binding of CD47s on cancer cells to SIRPs on surfaces of macrophages, binding to HER2s on cancer cells to inhibit uncontrolled cancer cell growth, and binding to FcRs on NK cells or macrophages.
2. The method for the recombinant fusion protein of claim 1, wherein each paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulatory protein (SIRP) at the N-terminus of the heavy chain constituting that paratope.
3. The method for the recombinant fusion protein of claim 1, wherein each paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulatory protein (SIRP) at the N-terminus of the light chain constituting that paratope.
4. The method for the recombinant fusion protein of claim 1, wherein one paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulatory protein (SIRP) at the N-terminus of the heavy chain constituting that paratope, and the other paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulatory protein (SIRP) at the N-terminus of the light chain constituting that paratope.
5. The method for the recombinant fusion protein of claim 1, wherein the signal-regulatory protein is SIRPα.
6. The method for the recombinant fusion protein of claim 5, wherein the extracellular Ig-like domain of the signal-regulatory protein is first extracellular Ig-like domain of signal-regulatory protein (SIRPαD1).
7. The method for the recombinant fusion protein of claim 1, wherein the linker is a peptide of 10 to 30 amino acid residues.
8. The method for the recombinant fusion protein of claim 7, wherein the linker is -(Gly-Gly-Gly-Gly-Ser)3-.
9. The method for the recombinant fusion protein of claim 1, wherein the Ig-like anti-HER2 antibody comprises two heavy chains each having an amino acid sequence with at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 6, and two light chains each having an amino acid sequence with at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 8.
10. The method for the recombinant fusion protein of claim 9, wherein each heavy chain has an amino acid sequence of SEQ ID NO: 6.
11. The method for the recombinant fusion protein of claim 9, wherein each light chain has an amino acid sequence of SEQ ID NO: 8.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0049] There are principally three different approaches to targeting two or more pharmacologies of tumor growth. Most commonly, patients can be given a cocktail of two or more different drugs. Although this option allows for maximal flexibility with respect to possible drug combinations and different dosages, it suffers from (a) potentially poor adherence to treatment by the patient because of the increased pill burden and the different dosing schedules for the individual drugs, (b) possible incompatibilities because of drug-drug interactions, and (c) increased risk of drug side effects. These problems may reduce the effectiveness of therapy and hamper the attainment of treatment goals particularly in the management of chronic diseases such as cancer.
[0050] The second approach relies on the use of fixed-dose combinations of drugs in a single dosage form. This approach reduces pill burden, resulting in improved patient compliance. The disadvantage of fixed-dose combinations is primarily the limited choice of possible dose ratios between the active ingredients, which makes it more difficult to properly titrate the individual patient to maximum efficacy with minimal adverse effects. In addition, different pharmacokinetic properties of the components in the combination might lead to a complex temporal mismatch in pharmacodynamic effects at the individual targets thereby compromising overall efficacy.
[0051] The third approach is the use of multifunctional drugs that combine two or more pharmacologies in a single entity. The design and validation of such multifunctional entities are more complex and require substantial investigation into the optimal ratio of target activities. Multifunctional molecules may also be amenable to fixed dose combination with other drugs thereby combining three or even four pharmacologies in a single pill to produce further increments in efficacy.
[0052] A recombinant bispecific or multi-specific protein against two or more targets is a multifunctional drug, which does not necessarily show superior anti-tumor activity compared to ordinary single antigen targeting antibodies. For example, as shown in the Example 8 below, a recombinant antibody comprising SIRPαD1, linked by a linker, to Erbitux (Cetuximab), an anti-EGFR antibody, had lower anti-tumor activity than Erbitux or SIRPαD1-Fc in the HT-29 or NCl-H1975 tumor model. Smart designs are needed for a recombinant protein to provide synergistic effects.
[0053] Through diligent experimentation, the present inventors have invented a novel recombinant fusion protein, which can attack tumors, via three mechanisms of actions, one to release the check on macrophages by SIRP-mediated inhibitory signals, one to control HER2 signaling mediated tumor/cancer cell proliferation, the third to stimulate cancer cell killings by NK cells and/or macrophages.
[0054] The recombinant fusion protein of the present disclosure comprises comprising an extracellular Ig-like domain of a signal-regulator protein (SIRP), linked via a linker, to a paratope of an Ig-like anti-HER2 antibody at the N-terminus of a heavy chain or a light chain constituting the paratope, wherein the recombinant fusion protein bind to CD47, HER2 and FcR simultaneously, i) binding to CD47s on cancer cells to block the interaction of CD47s with SIRPs on macrophages and thus releasing the check on macrophages by SIRP-mediated inhibitory signals; ii) binding to HER2 on cancer cells and thus inhibiting uncontrolled tumor/cancer cell growth; and iii) binding to FcRs on NK cells or macrophages to stimulate cancer cell killings by NK cells or macrophages. In an embodiment, either paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the heavy chain or the light chain constituting the paratope. In an embodiment, each paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the heavy chain or the light chain constituting that paratope. In one embodiment, each paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the heavy chain constituting that paratope. In one embodiment, each paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the light chain constituting that paratope. In one embodiment, one paratope of the Ig-like anti-HER2 antibody is linked to an extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the heavy chain constituting that paratope, and the other paratope is linked to an extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the light chain constituting that paratope. In some embodiment, one paratope of the anti-HER2 antibodies may be linked to two copies of the extracellular Ig-like domain of signal-regulator protein (SIRP) at the N-terminus of the heavy chain and light chain constituting that paratope.
[0055] The three main components contained in the fusion protein of the present disclosure are an extracellular Ig-like domain of a signal-regulator protein (SIRP), a linker, and an anti-HER2 antibody. A person of ordinary skills in the art will recognize that there are many design choices for selecting the above three components. Preferably, human-derived sequence is used in human cancer therapies, as the strong immunogenicity of the proteins or peptides from non-human animals may lead to allergy and other adverse effects. However, other animal proteins or peptides, humanized if appropriate, may also be used in the present disclosure based on different application purposes.
[0056] Any extracellular Ig-like domain of any SIPR (SIRPα, SIRPβ, and SIRPγ) capable of binding with CD47 may be selected for construction of the fusion protein. In one embodiment, the signal-regulatory protein in the recombinant fusion protein is SIRPα, and the extracellular Ig-like domain of the signal-regulatory protein is the first extracellular Ig-like domain of SIRPα (SIRPαD1).
[0057] In one embodiment, the recombinant fusion protein comprises SIRPαD1 having the nucleic acid sequence and amino acid sequence set forth in SEQ ID NOs: 1 and 2, respectively. In another embodiment, the SIRPαD1 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 2, wherein the SIRPαD1 can bind to CD47 on the cell surface of cancer/tumor cells and block the interaction of CD47 with SIRPs on the cell surfaces of macrophages.
[0058] Linkers serve primarily as a spacer between the extracellular Ig-like domain of SIRP and the N-terminus of the heavy or light chain of an anti-HER2 antibody. The linker may be made up of amino acids linked together by peptide bonds, preferably from 5 to 30 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. When two copies of the SIRP extracellular Ig-like domain are linked to one paratope of the anti-HER2 antibody at the N-terminus of the heavy chain and the light chain constituting that paratope, a relatively long linker, maybe of 10 or more, or even 15 or more amino acid resides in length, may be needed to avoid possible stereo hindrance. One or more of these amino acids may be glycosylated, as is understood by those of skill in the art. In one embodiment, the 5 to 30 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine and lysine. In one embodiment, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Exemplary linkers are polyglycines (particularly (Glys, (Gly).sub.8, poly(Gly-Ala), and polyalanines. One exemplary suitable linker as shown in the Examples below is poly(Gly-Ser), such as -(Gly-Gly-Gly-Gly-Ser).sub.3- (SEQ ID NO: 21).
[0059] Linkers may also be non-peptide linkers. For example, alkyl linkers such as —NH—, —(CH.sub.2)s-C(O)—, wherein s=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C.sub.1-4) lower acyl, halogen (e.g., CI, Br), CN, NH.sub.2, phenyl, etc.
[0060] Any anti-HER2 antibody, especially any Ig-like anti-HER2 antibody, may be used in the formation of the fusion protein of the present disclosure. The anti-HER2 antibody may be an isolated monoclonal antibody such as Trastuzumab and Margetuximab.
[0061] In some embodiments, the anti-HER2 antibody is an isolated monoclonal antibody comprising two heavy chains each having an amino acid sequence of SEQ ID NO: 6, and two light chains each having an amino acid sequence of SEQ ID NO: 8, which two may be encoded by nucleic acid sequences of SEQ ID NOs: 5 and 7, respectively. The Fab or paratope portion of the anti-HER2 antibody can bind to HER2 on the cell surfaces of cancer/tumor cells and thus prevent the occurring of uncontrolled growth of cancer/tumor cells, while the Fc portion of the anti-HER2 antibody can bind to FcRs on the cell surfaces of NK cells or macrophages to stimulate cancer cell killings by the NK cells or macrophages. In some embodiments, the heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 6, wherein the anti-HER2 antibody is able to bind to HER2 and prevent the occurring of uncontrolled growth of cancer/tumor cells, and is also able to bind to FcRs on the cell surfaces of NK cells or macrophages and thus activate the NK cells or macrophages for killing the cancer cells. In some embodiments, the light chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 8, wherein the anti-HER2 antibody is able to bind to HER2 and prevent occurring of uncontrolled growth of cancer/tumor cells.
[0062] As described above, one or two copies of the SIRP extracellular Ig-like domain especially SIRPαD1 can be linked to either or each paratope of the anti-HER2 at the N-terminal of the heavy chain and/or the light chain constituting the specific paratope.
[0063] Also, the present disclosure provides a polynucleotide molecule encoding the recombinant fusion protein and an expression vector expressing the recombinant bi-functional fusion protein. Examples of vectors include but are not limited to plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformation-competent artificial chromosomes (TACs), mammalian artificial chromosomes (MACs) and human artificial episomal chromosomes (HAECs).
[0064] The present disclosure provides host cells comprising the above expression vectors. The host cells may be transformed or transfected with the expression vectors. Suitable host cells include Escherichia coli, yeasts and other eukaryotes. Preferably, Escherichia coli, yeast or mammalian cell lines (such as COS or CHO) are used.
[0065] In another aspect, the present disclosure provides a pharmaceutical composition comprising the fusion protein of the present disclosure formulated together with a pharmaceutically acceptable adjuvant. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug. The pharmaceutical compositions of the disclosure also can be administered in a combination therapy with, for example, another immune-stimulatory agent, anti-cancer agent, or a vaccine.
[0066] The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.
[0067] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in injection. For example, the vehicle or carrier may be neutral buffered saline or saline mixed with serum albumin. Other exemplary pharmaceutical compositions comprise Tris buffers, or acetate buffers, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present disclosure, compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the therapeutic composition may be formulated as a lyophilizate using appropriate excipients such as sucrose.
[0068] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active molecule can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. 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, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
[0069] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.
[0070] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of active ingredient in combination with a pharmaceutically acceptable carrier.
[0071] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the fusion protein can be administered as a sustained release formulation, in which case less frequent administration is required.
[0072] For administration of the fusion protein, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 10 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration twice per week, once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Preferred dosage regimens for the fusion protein of the disclosure include 3 mg/kg body weight or 6 mg/kg body weight via intraperitoneal administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks; (vi) 6 mg/kg body weight, one dosage per week. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/ml and in some methods about 25-300 μg/ml.
[0073] A “therapeutically effective dosage” of a fusion protein of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of tumor-bearing subjects, a “therapeutically effective dosage” preferably inhibits tumor growth by at least about 40%, more preferably by at least about 60%, even more preferably by at least about 80%, and still more preferably by at least about 99% relative to untreated subjects. A therapeutically effective amount of a fusion protein of the present disclosure can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.
[0074] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0075] Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparatuses (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.
[0076] In certain embodiments, the fusion protein of the disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic fusion proteins of the disclosure cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331.
[0077] A gene therapy in vivo is also envisioned wherein a nucleic acid molecule encoding the recombinant fusion protein of the present disclosure, or a derivative thereof is introduced directly into the subject. For example, a nucleic acid sequence encoding a recombinant fusion protein of the present disclosure is introduced into target cells via local injection of a nucleic acid construct with or without an appropriate delivery vector, such as an adeno-associated virus vector. Alternative viral vectors include, but are not limited to, retroviruses, adenovirus, herpes simplex vims and papilloma virus vectors. Physical transfer of the virus vector may be achieved in vivo by local injection of the desired nucleic acid construct or other appropriate delivery vector containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or microparticle bombardment (gene-gun).
[0078] The compositions of the present disclosure may be used alone or in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects.
[0079] Another object of the present disclosure is to provide a method for preparing the above recombinant fusion protein and the pharmaceutical composition comprising the same. In one embodiment, the method comprises (1) providing an protein-encoding polynucleotide molecule; (2) constructing an expression vector comprising the polynucleotide molecule of (1); (3) transfecting or transforming suitable host cells with the expression vector of (2) and cultivating the host cells to express the protein; and (4) purifying the protein. The preparation may be carried out with well-known technologies by an ordinarily skilled artisan.
[0080] Another object of the present disclosure is to provide a method of treating cancer using the pharmaceutical composition of the present disclosure, comprising administrating an effective amount of the aforementioned pharmaceutical composition to the patients or subjects in need thereof. In one embodiment, the pharmaceutical composition is used to treat CD47 and/or HER2-overexpressing tumors or cancers, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-hodgkins lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, gastric cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer and renal cancer.
[0081] In one embodiment, the diseases related to over-expressions of CD47 and/or HER2 include but are not limited to Crohn's disease, allergic asthma, and rheumatoid arthritis.
[0082] The present disclosure is now further described with the non-limiting examples below.
EXAMPLES
[0083] In the examples below, IMM29 refers to a HER2-specific antibody. This antibody has two heavy chains each having an amino acid sequence of SEQ ID NO: 6, and two light chains each having an amino acid sequence of SEQ ID NO: 8, which two may be encoded by nucleic acid sequences of SEQ ID NOs: 5 and 7, respectively.
[0084] IMM2901 is a recombinant fusion protein capable of binding to CD47 and HER2, containing two SIRPαD1s each linked via a GS-linker, to IMM29 at the N-terminus of each heavy chain, wherein the SIRPαD1 has an nucleic acid sequence and amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the linker having an amino acid sequence of SEQ ID NO: 4, which can be encoded by the nucleic acid sequence of SEQ ID NO: 3.
[0085] IMM2902 is also a recombinant fusion protein capable of binding to CD47 and HER2, and differs from IMM2901 in that each SIRPαD1 is linked via a GS-linker, to IMM29 at the N-terminus of each light chain.
[0086] IMM01 is a fusion protein capable of binding to CD47, consisting of SIRPαD1 linked to an Fc fragment, which was described in WO2016169261. The nucleic acid sequence and amino acid sequence of this fusion protein are set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
[0087] IMM0404 is a recombinant fusion protein, containing two SIRPαD1s each linked via a GS-linker, to an anti-EGFR antibody at the N-terminus of each heavy chain. The SIRPαD1-GS-linker-anti-EGFR heavy chain has a nucleic acid sequence and amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 16, respectively. The light chain of the anti-EGFR antibody has an amino acid sequence of SEQ ID NO: 18, which may be encoded by nucleic acid sequences of SEQ ID NO: 17.
[0088] The structures of these proteins can be found in
Example 1. Construction of Vectors Expressing IMM29, IMM2901, IMM2902, IMM01 and IMM0404
1.1 IMM29
[0089] The full-length coding sequence of IMM29 was designed artificially. Specifically, the coding sequences of both the heavy chain and the light chain variable regions were derived from Herceptin (Trastuzumab). 57 nucleotides encoding the signal peptide of mouse IgG1 heavy chain (SEQ ID NO.: 19) were added to the 5′ end of the heavy chain-coding sequence (SEQ ID NO.: 5) or the light chain-coding sequence (SEQ ID NO.:7), and a Kozak sequence (SEQ ID NO.: 20: GCCGCCACC) was added to the 5′ end of the signal peptide sequence. Then, HindIII and NheI restriction sites were added to the 5′ and 3′ ends of the resulting heavy chain sequence, and HindIII and the XbaI restriction sites were added to the 5′ and 3′ ends of the resulting light chain sequence. The two resulting sequences were synthesized by Genscript (ID #: T84300 (heavy chain); T85555 (light chain)) and subcloned, respectively, into the pMac-H and pMac-L vectors.
1.2 IMM2901
[0090] The expression vector for the light chain of IMM2901 is identical to that of IMM29. For the heavy chain vector construction, the coding sequence of the first extracellular domain of SIRPα (SIRPαD1) (SEQ ID NO.:1) was linked through a GS-linker (SEQ ID NO.:3) to the N terminal of the heavy chain coding sequence of IMM29 (SEQ ID NO.:5) (totally SEQ ID NO.: 9). 57 nucleotides encoding the signal peptide of mouse IgG1 heavy chain (SEQ ID NO.:19) were added to the 5′ end of SIRPαD1-coding sequence, and a Kozak sequence (SEQ ID NO.: 20: GCCGCCACC) was added to the 5′ end of the signal peptide sequence. Lastly, HindIII and NheI restriction sites were added to the 5′ and 3′ ends of the resulting sequence, respectively. The resulting sequence was synthesized by Convenience Biology (ID #: Y0000506-1-A10863) and subcloned into the pMac-H vector.
1.3 IMM2902
[0091] The expression vector for the heavy chain of IMM2902 is identical to that of IMM29. For the light chain vector construction, the coding sequence of the first extracellular domain of SIRPα (SIRPαD1) (SEQ ID NO.:1) was linked through a GS-linker (SEQ ID NO.:3) to the N terminal of the light chain coding sequence of IMM29 (SEQ ID NO.:7) (totally SEQ ID NO.: 11). 57 nucleotides encoding the signal peptide of mouse IgG1 heavy chain (SEQ ID NO.:19) were added to the 5′ end of SIRPαD1-coding sequence, and a Kozak sequence (SEQ ID NO.: 20: GCCGCCACC) was added to the 5′ end of the signal peptide sequence. Lastly, HindIII and XbaI restriction sites were added to the 5′ and 3′ ends of the resulting sequence, respectively. The resulting sequence was synthesized by Convenience Biology (ID #: Y0000506-2-A10868) and subcloned into the pMac-L vector.
1.4 IMM01
[0092] The expression cassette of SIRPαD1-Fc was designed by sequentially connecting a Kozak sequence (SEQ ID NO.: 20: GCCGCCACC) with the coding sequence of the signal peptide (SEQ ID NO.:19) and SIRPαD1-Fc (SEQ ID NO.:13). HindIII and EcoRI restriction sites were respectively added to the 5′ and 3′ ends of the resulting sequence, which was synthesized by Convenience Biology (ID #: CN1418-F9043) and subcloned into the pMac-Fc vector. 1.5 IMM0404
[0093] The expression cassette for the light chain of IMM0404 was designed by sequentially connecting a Kozak sequence (SEQ ID NO.: 20: GCCGCCACC) with the coding sequence of the signal peptide (SEQ ID NO.:19) and the light chain of an anti-EGFR antibody (Erbitux (Cetuximab)) (SEQ ID NO.:17). HindIII and XbaI restriction sites were respectively added to the 5′ and 3′ ends of the resulting sequence, which was synthesized by Convenience Biology (ID #: NJ0719028J_A6315) and subcloned into the pMac-L vector. For the heavy chain vector construction, the coding sequence of the first extracellular domain of SIRPα (SIRPαD1) was linked through a GS-linker to the N terminal of the heavy chain coding sequence of an EGFR-specific antibody, the SIRPαD1-GS-linker-heavy chain was encoded by SEQ ID NO.:15. 57 nucleotides encoding the signal peptide of mouse IgG1 heavy chain (SEQ ID NO.:19) were added to the 5′ end of SIRPαD1-GS-linker-heavy chain (SEQ ID NO.: 15), and a Kozak sequence (SEQ ID NO.: 20: GCCGCCACC) was added to the 5′ end of the signal peptide sequence. Lastly, HindIII and NheI restriction sites were added to the 5′ and 3′ ends of the resulting sequence, which was synthesized by Genscript (ID #: M17025) and subcloned into the pMac-H vector.
Example 2. Protein Expression and Purification
[0094] To manufacture the desired proteins, the expression vectors of Example 1 were electroporated into Chinese Hamster Ovary (CHO) cells (ATCC, Cat #CCL-61) which were subjected to several rounds of pressure selection of neomycin. The selected stable cells were adapted to a serum-free Balan CD CHO Growth A medium (Irvine Scientific, Cat #941 20). For protein expression, cells were seeded in a 3 L bioreactor and cultured in a fed-batch process. When the cell viability dropped to ˜80%, reaction in the bioreactor was terminated, the cell culture supernatant was harvested and subjected to protein purification by affinity chromatography. The purity of recombinant protein was above 95%, and the content of endotoxin was below 0.5 U/g.
Example 3. IMM2901 and IMM2902 Bound to CD47 or HER2
[0095] CD47 or HER2 binding capacities of the recombinant proteins were measured by the enzyme-linked immunosorbent assay (ELISA). Recombinant Human CD47 (Lot #LC10DE2004, Sino Biologicals) and ErbB2 (Lot #LC11MC0201, Sino Biologicals) were, respectively, prepared in coating buffer (Product code: 1001329288 C3041-100CAP, Sigma-Aldrich Co.) and transferred to the ELISA plates (Cat #442404, Nunc™) at 50 ng/well. The plates were placed in 4° C. refrigerator overnight. When assays were performed, plates were washed for three times with PBS containing 0.05% of Tween-20 (PBS-T) before the titrated proteins were added, and the plates were incubated at room temperature for 1 hour. The plates were washed again for 5 times with PBS-T, and then HRP-Rabbit Anti-Human IgG Fc (Cat #:309-036-008, Jackson ImmunoResearch Lab) was added to the plates and incubated at room temperature for one hour. After the plates were washed for 5 times with PBS-T, and substrates were added to the plates which were read in a plate reader after the color changing was stopped by 1N H.sub.2SO.sub.4.
[0096] IMM2901 and IMM2902 bound to CD47 with an EC.sub.50 value of 0.1903 nM and 0.3894, respectively (
Example 4. IMM2902 Activated Phagocytosis of HL-60
[0097] Mouse macrophage cell line Ana-1 was seeded in a 96-well cell culture plate, 1×10.sup.5 cells per well, and cultured for 16-18 hours at 37° C. and 5% CO.sub.2. Target cells (HL-60) were labeled with CF SE, and then incubated with serially diluted IMM2902, or control proteins for 45 minutes. The target cell solutions containing the test proteins were transferred to the plate containing Ana-1 cells, the ratio of the number of Ana-1 cells to HL-60 cells was 1:3. The mixture was cultured for 2 hours at a cell culture incubator and then subject to FACS analysis for density of CFSE in Ana-1 cells.
[0098] As shown in
Example 5. IMM2902 had High Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
[0099] CFSE-labeled BT-474 cells (used as target cells) were mixed with NK92MI cells (effector cells) stably expressing FcγRIIIa at a 1:2 ratio, and the mixed cells were cultured for 4 hours at 37° C. under 5% CO.sub.2 in the presence of serially diluted IMM2902 or control proteins. Then 5 g/ml propidium iodide (PI) (Sigma, Cat#P4170) was added to the cell culture at a concentration of 5 μg/ml, and the cell culture was subjected to FACS analysis for PI signals. Percentage of cell lysis mediated by ADCC was calculated based on the following formula:
% Lysis=(% PI Positive Cell with IMM2902 or control proteins−% PI Positive Cell with negative control protein)/(100−% PI Positive Cell with negative control protein)*100
[0100] As shown in
Example 6. IMM2902 Induced HER2 Internalization
[0101] 1×106 of BT-474 cells in 200 L of DMEM medium containing 5% of FBS were seeded in a 96-well cell culture plate and incubated at 37° C. and 5% CO.sub.2 overnight. On the next day, the plate was taken out of the incubator and the medium was replaced with fresh medium containing titrated proteins. The plate was incubated for further 4 hours in the cell culture incubator before the cells were washed and stained with FITC-conjugated antibody specific for the Fc portion of human IgG. Percentage of HER2 receptor internalized was calculated based on the formula below:
Internalization Ratio=(1−MFI/MFIt=0)*100%
[0102] MFI: mean fluorescence intensity
[0103] According to
Example 7. IMM2902 had Good Anti-Tumor Effect
[0104] BT-474 human breast cancer cells (ATCC® Number: HTB-20; Lot number: 63087043) were cultured in a DMEM medium containing 10% FBS at 37° C. and 5% CO.sub.2.
[0105] Cells were collected and re-suspended in a serum-free DMEM medium, 1×10.sup.8/mL. The medium was added with and mixed with Matrige at a volume ratio of 1:1 and then placed on ice for use.
[0106] Fifty-five nude mice were injected subcutaneously with BT-474 cells, 1×10.sup.7 cells per mouse, at the right flank. These mice were given intramuscular injections of estrogens one week prior to tumor cell injection till the end of the test, three times a week (every Monday, Wednesday, and Friday), to keep growth of the estrogen-dependent tumor.
[0107] When tumor volume reached 100-150 mm.sup.3, 36 mice were randomly allocated into 6 groups with 6 mice in each group. Mice were respectively treated, twice a week, through intraperitoneal injection with PBS, IMM01 (3.0 mg/kg), Herceptin (5.0 mg/kg), IMM29 (5.0 mg/kg), IMM2909 (6.0 mg/kg), and IMM01+IMM29 (3.0 mg/kg+5.0 mg/kg), for 3 weeks. Totally six treatments were given. The day upon first dosing was defined as Day 0. Tumor volume and body weight were measured twice a week.
[0108] During treatments, if a mouse lost 15% or more of body weight, drug administration would be stopped until the weight loss became 10% or less. Animals were sacrificed when the average tumor volume in any group exceeded 2000 mm.sup.3 or the experiment was completed.
[0109] The tumor volume (V) was calculated as (length×width2)/2. Tumor growth inhibition rate (TGI) was calculated by the formula: Tumor growth inhibition rate=(1−tumor volume change in administration group/tumor volume change in control group)×100%. Dunnett's multi-comparison test was used to calculate group differences.
TABLE-US-00001 TABLE 1 Anti-tumor effect of IMM2902 and other antibodies Dose Group Drug Animal# (mg/kg) Treatment TGI* 1 PBS 6 N/A i.p, b.i.w × 3 2 IMM01 6 3.0 i.p, b.i.w × 3 25.46% 3 Herceptin 6 5.0 i.p, b.i.w × 3 86.95% 4 IMM29 6 5.0 i.p, b.i.w × 3 104.23% 5 IMM2902 6 6.0 i.p, b.i.w × 3 115.28% 6 IMM01 + 6 3.0 + 5.0 i.p, b.i.w × 3 110.88% IMM29
[0110] Group 5 had a tumor growth inhibition rate (TGI) of 115.28%, higher than those of other groups, as shown in Table 1 above and
Example 8. IMM0404's Anti-Tumor Activity in HT-29 or NCl-H1975 Xenograft Model
[0111] 8.1 HT-29 Xenograft Model
[0112] HT-29 human colon cancer cells were cultured in the McCoy's 5A medium containing 10% FBS at 37° C. and 5% CO.sub.2.
[0113] Cells at the logarithmic phase were collected and re-suspended in 1×PBS. The suspension was added with and mixed with Matrige at a volume ratio of 1:1, and the mixture contained 3×10.sup.7 cells per mL.
[0114] Forty mice were injected subcutaneously with HT-29 cells, 3×10.sup.6 cells per mouse, at the right flank. When tumor volume reached 100-200 mm.sup.3, these animals were randomly allocated into 5 groups with 8 mice in each group. Mice were respectively treated, once per week, through intraperitoneal injection with PBS, IMM01 (1.2 mg/kg), Erbitux (2.0 mg/kg), IMM0404 (2.7 mg/kg), and IMM01+Erbitux (1.2 mg/kg+2.0 mg/kg), for 4 weeks. Totally four treatments were given. The day upon first dosing was defined as Day 0. Tumor volume and body weight were measured twice a week.
[0115] The tumor volume (V) was calculated as (length×width.sup.2)/2. Tumor growth inhibition rate (TGI) was calculated by the formula: Tumor growth inhibition rate=(1−tumor volume change in administration group/tumor volume change in control group)×100%. The student test was used to calculate group differences.
TABLE-US-00002 TABLE 2 Anti-tumor effect of IMM0404 and other antibodies Dose Group Drug Animal# (mg/kg) Treatment TGI* 1 PBS 8 N/A i.p, q.w. × 4 2 IMM01 8 1.2 i.p, q.w. × 4 32.79% 3 Eribitux 8 2.0 i.p, q.w. × 4 40.00% 4 IMM0404 8 2.7 i.p, q.w. × 4 18.48% 5 IMM01 + 8 1.2 + 2.0 i.p, q.w. × 4 33.04% Eribitux
[0116] It can be seen from Table 2 and
[0117] 8.2 NCI-H1975 Xenograft Model
[0118] NCI-H1975 non-small cell lung cancer cells were cultured in the RPMI-1640 medium containing 10% FBS (GIBCO, US) at 37° C. and 5% CO.sub.2.
[0119] Cells at the logarithmic phase were collected and re-suspended in 1×PBS, 1×10.sup.7 cells per mL.
[0120] Forty SCID mice were injected subcutaneously with NCI-H1975 cells, 1×10.sup.6 cells per mouse, at the right flank. When tumor volume reached 100-200 mm.sup.3, these animals were randomly allocated into 5 groups with 8 mice in each group. Mice were respectively treated, once per week, through intraperitoneal injection with PBS, IMM01 (2.7 mg/kg), Erbitux (5.0 mg/kg), IMM0404 (6.0 mg/kg), and IMM01+Erbitux (2.7 mg/kg+5.0 mg/kg), for 3 weeks. Totally three treatments were given. The day upon first dosing was defined as Day 0. Tumor volume and body weight were measured twice a week.
[0121] The tumor volume (V) was calculated as (length×width.sup.2)/2. Tumor growth inhibition rate (TGI) was calculated by the formula: Tumor growth inhibition rate=(1−tumor volume change in administration group/tumor volume change in control group)×100%. The student test was used to calculate group differences.
TABLE-US-00003 TABLE 3 Anti-tumor effect of IMM0404 and other antibodies Dose Group Drug Animal# (mg/kg) Treatment TGI* 1 PBS 8 N/A i.p, q.w. × 3 2 IMM01 8 2.7 i.p, q.w. × 3 49.49% 3 Eribitux 8 5.0 i.p, q.w. × 3 85.69% 4 IMM0404 8 6.0 i.p, q.w. × 3 68.77% 5 IMM01 + 8 2.7 + 5.0 i.p, q.w. × 3 76.03% Eribitux
[0122] It can be seen from Table 3 and
[0123] The data in this Example suggested that the bispecific antibodies do not necessarily show superior efficacy compared to the single antigen targeting antibodies.
Example 9. Exemplary Recombinant Fusion Protein Bound CD47.SUP.+.HER2.SUP.+.Tumor Cells
[0124] Briefly, 5×10.sup.5/ml NCl-N87 cells (a gastric cancer cell line, Cat #TCHu130, Cell bank of Chinese Academy of Sciences) in 50 μl 0.5% BSA-PBS were seeded onto 96-well plates. The plates were added with 50 μl serially diluted IMM01, Herceptin®, IMM2902 and hIgG1-Fc, respectively, and incubated for 45 minutes at 4° C. The plates were washed using 0.5% BSA-PBS to remove unbound proteins, added with the secondary antibody FITC anti-human IgG Fc (1:500), and detected for the FITC fluorescence signals by flow cytometry.
[0125] The assay was also performed with 5×10.sup.5/ml SKO-V3 cells (an ovarian cancer cell line, Cat #TCHu185, Cell bank of Chinese Academy of Sciences) in 50 μl 0.5% BSA-PBS, following the protocol above.
[0126] As shown in
Example 10. Exemplary Recombinant Fusion Protein Induced Antibody-Dependent Cell Mediated Cytotoxicity (ADCC) Against CD47.SUP.+.HER2.SUP.+.Tumor Cells
[0127] FcR-TANK cells (ImmuneOnco-engineered cell line, NK cell line overexpress Fc gamma RIIIA), as the effector cells, in 100 μl medium at the density of 5×10.sup.5/ml, were mixed respectively with NCl-N87 cells and SK-OV-3 cells, as the target cells, in 50 μl medium at 5×10.sup.5/ml, in 96-well plates. Then, the cell mixtures were added and incubated with 50 μl serially diluted IMM2902, Herceptin®, IMM01 and hIgG1-Fc, respectively, at 37° C. with 5% CO.sub.2 overnight. The plates were added with 20 μl CCK-8 and incubated at 37° C. with 5% CO.sub.2 for 2 hours, and determined for absorbance at 450 nm. The assay was done in duplicate.
[0128] ADCC % was calculated using the following formula, wherein OD450(Sample), OD450(FcR-TANK) and OD450(Target cell+FcR-TANK) referred to OD450 value of the plate well with the test article, OD450 value of the well with FcR-TANK cells alone, and OD450 value of the well with target cells and FcR-TANK cells alone, respectively.
[0129] The results were shown in
[0130] The assay was also performed on the NCl-N87 cells as the target cells and FcR_158V-TANK or FcR_158F-TANK cells as the effector cells, following the protocol above. The FcR_158V-TANK cells expressed on their surfaces Fc gamma RIIIA having 158V mutation while the FcR_158F-TANK cells expressed Fc gamma RIIIA with 158F mutation.
[0131] The results were shown in
Example 11. Recombinant Fusion Protein Induced Antibody-Dependent Cellular Phagocytosis (ADCP) Against CD47.SUP.+.HER2.SUP.+.Tumor Cells
[0132] THP-1 cells (macrophages) were centrifuged at 1000 rpm for 5 min, collected, washed with the 1640 complete culture medium (1640+10% FBS+1% PS), and cultured at the density of 4×10.sup.5/ml with 200 ng/ml PMA. Then, 100 μl THP-1 cells were seeded onto each well of 96-well plates, and cultured in a cell incubator for 48 hours.
[0133] CFSE at 1 mM was 1:250 diluted into the cell culture with SKO-V3 cells at 1×10.sup.6/ml to label the tumor cells for 30 min in the cell incubator. The CFSE-labeled SKO-V3 cells were washed with the culture medium and adjusted to the cell density of 1×10.sup.6/ml.
[0134] The supernatants were removed from the plates where the THP-1 cells were cultured. The plates were washed with 100 μl PBS, and added with 100 μl serially diluted IMM2902, Herceptin®, IMM01+Herceptin®, IMM01 or hIgG1-Fc (4-fold dilution starting at 2 nM), and 100 CFSE-labeled SKO-V3, and incubated in the cell incubator for 2 hours.
[0135] The plates were washed with 200 μl PBS for three times, incubated with 50 μl trypsin (1:5 dilution in PBS) for 3 min, added with 150 μl PBS, and subject to a flow cytometer to detect fluorescence signals from the THP-1 cells.
[0136] As can be seen from
Example 12. Recombinant Fusion Protein Showed Potent In Vivo Anti-Tumor Activity
[0137] SNU-1 cells (a gastric cancer cell line, Cell bank of Chinese Academy of Sciences, Cat #TCHu230) were cultured in RPMI1640 medium with 10% FBS and 1% Penicillin-Streptomycin in a cell incubator at 37° C. and 5% CO.sub.2.
[0138] Cells were collected and re-suspended in PBS. PBS, 100 μl in volume, containing 10×10.sup.6 SNU-1 cells, was added and mixed with 100 μl Matrige.
[0139] CB17-SCID female mice, 6-8 weeks old, were injected subcutaneously with the SNU-1 cells as prepared above at the right axilla.
[0140] When tumor sizes reached about 142 mm.sup.3, the mice were randomly allocated into 5 groups with 6 mice in each group, and this day was designated as Day 0. Mice were intraperitoneally administered with DPBS, Inetetamab (a commercially available anti-HER2 antibody), and IMM2902 respectively following the dosing regime in Table 4.
TABLE-US-00004 TABLE 4 Dosing regime Dose Dosing volume Group Drug (mg/kg) (μl/g body weight) Treatment 1 DPBS — 10 IP, BIW × 4 W 2 Inetetamab 8 10 IP, BIW × 4 W 3 IMM2902 2 10 IP, BIW × 4 W 4 IMM2902 6 10 IP, BIW × 4 W 5 IMM2902 18 10 IP, BIW × 4 W
[0141] The mice were observed for their daily physical activity, and measured for body weights and tumor sizes twice a week. The tumor volume (V) was calculated as (length×width.sup.2)/2. Tumor growth inhibition rate (TGI) and relative tumor proliferation rate (T/C, %) were calculated by the following formulae.
Tumor growth inhibition (TGI,%)=(1−tumor volume change in drug treatment group/tumor volume change in vehicle control group)×100%.
T/C (%)=RTV in drug treatment group/RTV in vehicle control group×100%,
RTV=Vt/V0, Vt and V0 referred to the average tumor size at Day t and the average tumor size at Day 0, respectively.
[0142] Animal deaths were observed on Day 28 and then on, due to tumor growth. The data at Day 28 were analyzed using t-test in SPSS, and group differences were deemed statistically significant when the p-value was 0.05 or lower.
TABLE-US-00005 TABLE 5 Anti-tumor efficacy of IMM2902 and Inetetamab average tumor size at Day 28 T/C TGI Group (mm.sup.3).sup.a (%) (%) p-value .sup.b 1 1257.91 ± 157.25 — — — 2 800.26 ± 126.69 63.66 41.02 0.047 3 655.51 ± 80.95 52.44 53.92 0.007 4 294.85 ± 56.61 23.46 86.33 0.001 5 239.89 ± 57.67 19.17 91.20 0.001 .sup.amean ± SEM; .sup.b compared to Group 1
[0143] The results were shown in
Example 13. Recombinant Fusion Protein Showed Higher In Vivo Anti-Tumor Activity than Monospecific Counterparts and their Combination
[0144] NCI-N87 cells (a gastric cancer cell line, Cat#TCHu130, Cell bank of Chinese Academy of Sciences) were cultured in RPMI1640 medium with 10% FBS in a cell incubator at 37° C. and 5% CO.sub.2.
[0145] Cells at the log phase were collected and re-suspended in RPMI1640 medium. The RPMI1640 medium was mixed with Matrige at 1:1 volume ratio, with the final cell density at 8×10.sup.7/ml.
[0146] CB17-SCID female mice were injected subcutaneously with the NCI-N87 cells as prepared above at the right side of the back, 8×10.sup.6 cells in 0.1 ml medium per mouse.
[0147] When tumor sizes reached about 121 mm.sup.3, the mice were randomly allocated into 7 groups with 10 mice in each group, and this day was designated as Day 0. Mice were intraperitoneally administered with PBS, IMM01, Herceptin®, IMM2902 and IMM01+Herceptin®, respectively, following the dosing regime in Table 6.
TABLE-US-00006 TABLE 6 Dosing regime Dosing Drug volume concen- Dose (μl/g body tration Group Drug (mg/kg) weight) (mg/ml) Treatment 1 PBS NA 10 NA IP, BIW × 4 W 2 IMM01 1.5 10 0.15 IP, BIW × 4 W 3 Herceptin ® 3 10 0.3 IP, BIW × 4 W 4 IMM2902 1 10 0.1 IP, BIW × 4 W 5 IMM2902 3.5 10 0.35 IP, BIW × 4 W 6 IMM2902 10 10 1 IP, BIW × 4 W 7 IMM01 0.5 5 0.1 IP, BIW × 4 W Herceptin ® 3 5 0.6 IP, BIW × 4 W
[0148] The mice were observed for their daily physical activity, and measured for body weights and tumor sizes twice a week. The tumor volume (V) was calculated as (length×width.sup.2)/2. Tumor growth inhibition rate (TGI), relative tumor proliferation rate (T/C, %), and animal body weight change (BWC, %) were calculated by the following formulae.
Tumor growth inhibition (TGI,%)=(average tumor size in vehicle control group−average tumor size in drug treatment group)/average tumor size in vehicle control group×100%
T/C (%)=RTV in drug treatment group/RTV in vehicle control group×100%,
wherein RTV (relative tumor volume)=Vt/V0, Vt and V0 referred to the average tumor size at Day t and the average tumor size at Day 0, respectively.
Animal body weight change (BWC,%)=body weight at Day t−body weight at Day 0)/body weight at Day 0×100%
[0149] The data were analyzed using the two tailed t-test, and group differences were deemed statistically significant when the p-value was 0.05 or lower, and highly statistically significant when the p-value was 0.01 or lower.
[0150] The results were shown in Table 7 and
TABLE-US-00007 TABLE 7 Anti-tumor efficacy of IMM2902, IMM01, and Herceptin ® Tumor size (mm.sup.3, Mean ± TGI (%)- Dose SEM) Tumor size T/C (%) Group Drug (mg/kg) Day 0 Day 35 Day 35 Day 35 1 PBS NA 121.08 ± 4.2 1152.37 ± 53.98 NA NA 2 IMM01 1.5 121.56 ± 4.31 786.78 ± 71.13** 31.73% 67.89% 3 Herceptin ® 3 121.88 ± 4.07 242.82 ± 29.62*** 78.93% 20.88% 4 IMM2902 1 121.61 ± 4.16 175.75 ± 29.66*** 84.75% 15.42% 5 IMM2902 3.5 121.16 ± 3.81 8.49 ± 4.41*** 99.26% 0.63% 6 IMM2902 10 121.07 ± 3.75 2.12 ± 2.12*** 99.82% 0.21% 7 IMM01 0.5 120.76 ± 3.6 258.47 ± 36.92*** 77.57% 22.56% Herceptin ® 3
[0151] While the invention has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents, as may be included within the spirit and scope of the appended claims. All referenced cited herein are further incorporated by reference in their entirety.
[0152] Sequences in the present application are summarized below.
TABLE-US-00008 First extracellular Ig-like domain of SIRPalpha SEQ ID NO: 1 GAGGAGGAGC TGCAGGTGAT TCAGCCTGAC AAGTCCGTAT CAGTTGCAGC TGGAGAGTCG GCCATTCTGC ACTGCACTGT GACCTCCCTG ATCCCTGTGG GGCCCATCCA GTGGTTCAGA GGAGCTGGAC CAGCCCGGGA ATTAATCTAC AATCAAAAAG AAGGCCACTT CCCCCGGGTA ACAACTGTTT CAGAGTCCAC AAAGAGAGAA AACATGGACT TTTCCATCAG CATCAGTGCC ATCACCCCAG CAGATGCCGG CACCTACTAC TGTGTGAAGT TCCGGAAAGG GAGCCCTGAC ACGGAGTTTA AGTCTGGAGC AGGCACTGAG CTGTCTGTGC GTGCCAAACC CTCTGCCCCC GTGGTATCGG GCCCT 375 First extracellular Ig-like domain of SIRPalpha SEQ ID NO: 2 EEELQVIQPD KSVSVAAGES AILHCTVTSL IPVGPIQWFR GAGPARELIY NQKEGHFPRV TTVSESTKRE NMDFSISISA ITPADAGTYY CVKFRKGSPD TEFKSGAGTE LSVRAKPSAP WSGP 125 Linker SEQ ID NO: 3 GGCGG CGGTGGGAGC GGCGGCGGTG GGAGCGGCGG CGGGGGCTCG 45 Linker SEQ ID NO: 4 GGGGSGGGGS GGGGS 15 Heavy chain of anti-HER2 antibody SEQ ID NO: 5 GAGGTGCAGC TGGTCGAGAG CGGCGGGGGC CTCGTGCAGC CGGGCGGGTC GCTGCGGCTG AGCTGCGCCG CGAGCGGGTT CAACATCAAG GACACCTACA TCCACTGGGT GCGCCAGGCC CCCGGCAAGG GCCTCGAGTG GGTCGCCCGG ATCTACCCCA CGAACGGGTA CACCCGCTAC GCCGACAGCG TGAAGGGCCG GTTCACCATC AGCGCGGACA CCTCGAAGAA CACGGCCTAC CTGCAGATGA ACAGCCTGCG CGCCGAGGAC ACCGCCGTGT ACTACTGCAG CCGGTGGGGC GGCGACGGGT TCTACGCCAT GGACTACTGG GGGCAGGGCA CCCTCGTCAC CGTGAGCAGC GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAG AGTTGAGCCC AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTGAACT CCTGGGGGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG TATGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC GCCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AAGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGCCGC AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGAGGAG ATGACCAAGA ACCAAGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAT TCCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGCAAA TGA 1353 Heavy chain of anti-HER2 antibody SEQ ID NO: 6 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN ATYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIAATIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 Light chain of anti-HER2 antibody SEQ ID NO: 7 GACATCCAGA TGACCCAGAG CCCGTCGAGC CTGAGCGCCA GCGTGGGCGA CCGGGTCACG ATCACCTGCC GCGCGAGCCA GGACGTGAAC ACCGCCGTGG CCTGGTACCA GCAGAAGCCC GGGAAGGCCC CCAAGCTCCT GATCTACTCG GCGAGCTTCC TGTACAGCGG CGTCCCCAGC CGGTTCAGCG GGTCGCGCAG CGGCACCGAC TTCACGCTCA CCATCAGCAG CCTGCAGCCG GAGGACTTCG CCACCTACTA CTGCCAGCAG CACTACACCA CGCCCCCCAC CTTCGGGCAG GGCACCAAGG TGGAGATCAA GCGAACTGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG 645 Light chain of anti-HER2 antibody SEQ ID NO: 8 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SIRP alpha Dl-Linker-anti-HER2 heavy chain SEQ ID NO: 9 GAGGAGGAGC TGCAGGTGAT TCAGCCTGAC AAGTCCGTAT CAGTTGCAGC TGGAGAGTCG GCCATTCTGC ACTGCACTGT GACCTCCCTG ATCCCTGTGG GGCCCATCCA GTGGTTCAGA GGAGCTGGAC CAGCCCGGGA ATTAATCTAC AATCAAAAAG AAGGCCACTT CCCCCGGGTA ACAACTGTTT CAGAGTCCAC AAAGAGAGAA AACATGGACT TTTCCATCAG CATCAGTGCC ATCACCCCAG CAGATGCCGG CACCTACTAC TGTGTGAAGT TCCGGAAAGG GAGCCCTGAC ACGGAGTTTA AGTCTGGAGC AGGCACTGAG CTGTCTGTGC GTGCCAAACC CTCTGCCCCC GTGGTATCGG GCCCTGGCGG CGGTGGGAGC GGCGGCGGTG GGAGCGGCGG CGGGGGCTCG GAGGTGCAGC TGGTCGAGAG CGGCGGGGGC CTCGTGCAGC CGGGCGGGTC GCTGCGGCTG AGCTGCGCCG CGAGCGGGTT CAACATCAAG GACACCTACA TCCACTGGGT GCGCCAGGCC CCCGGCAAGG GCCTCGAGTG GGTCGCCCGG ATCTACCCCA CGAACGGGTA CACCCGCTAC GCCGACAGCG TGAAGGGCCG GTTCACCATC AGCGCGGACA CCTCGAAGAA CACGGCCTAC CTGCAGATGA ACAGCCTGCG CGCCGAGGAC ACCGCCGTGT ACTACTGCAG CCGGTGGGGC GGCGACGGGT TCTACGCCAT GGACTACTGG GGGCAGGGCA CCCTCGTCAC CGTGAGCAGC GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAG AGTTGAGCCC AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTGAACT CCTGGGGGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG TATGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC GCCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AAGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGCCGC AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGAGGAG ATGACCAAGA ACCAAGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAT TCCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGCAAA TGA 1773 SIRP alpha D1-Linker-anti-HER2 heavy chain SEQ ID NO: 10 EEELQVIQPD KSVSVAAGES AILHCTVTSL IPVGPIQWFR GAGPARELIY NQKEGHFPRV TTVSESTKRE NMDFSISISA ITPADAGTYY CVKFRKGSPD TEFKSGAGTE LSVRAKPSAP VVSGPGGGGS GGGGSGGGGS EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN ATYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIAATIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 590 SIRP alpha Dl-Linker-anti-HER2 light chain SEQ ID NO: 11 GAGGAGGAGC TGCAGGTGAT TCAGCCTGAC AAGTCCGTAT CAGTTGCAGC TGGAGAGTCG GCCATTCTGC ACTGCACTGT GACCTCCCTG ATCCCTGTGG GGCCCATCCA GTGGTTCAGA GGAGCTGGAC CAGCCCGGGA ATTAATCTAC AATCAAAAAG AAGGCCACTT CCCCCGGGTA ACAACTGTTT CAGAGTCCAC AAAGAGAGAA AACATGGACT TTTCCATCAG CATCAGTGCC ATCACCCCAG CAGATGCCGG CACCTACTAC TGTGTGAAGT TCCGGAAAGG GAGCCCTGAC ACGGAGTTTA AGTCTGGAGC AGGCACTGAG CTGTCTGTGC GTGCCAAACC CTCTGCCCCC GTGGTATCGG GCCCTGGCGG CGGTGGGAGC GGCGGCGGTG GGAGCGGCGG CGGGGGCTCG GACATCCAGA TGACCCAGAG CCCGTCGAGC CTGAGCGCCA GCGTGGGCGA CCGGGTCACG ATCACCTGCC GCGCGAGCCA GGACGTGAAC ACCGCCGTGG CCTGGTACCA GCAGAAGCCC GGGAAGGCCC CCAAGCTCCT GATCTACTCG GCGAGCTTCC TGTACAGCGG CGTCCCCAGC CGGTTCAGCG GGTCGCGCAG CGGCACCGAC TTCACGCTCA CCATCAGCAG CCTGCAGCCG GAGGACTTCG CCACCTACTA CTGCCAGCAG CACTACACCA CGCCCCCCAC CTTCGGGCAG GGCACCAAGG TGGAGATCAA GCGAACTGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG 1065 SIRP alpha D1-Linker-anti-HER2 light chain SEQ ID NO: 12 EEELQVIQPD KSVSVAAGES AILHCTVTSL IPVGPIQWFR GAGPARELIY NQKEGHFPRV TTVSESTKRE NMDFSISISA ITPADAGTYY CVKFRKGSPD TEFKSGAGTE LSVRAKPSAP VVSGPGGGGS GGGGSGGGGS DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 354 SIRP alpha Dl-Fc SEQ ID NO: 13 GAGGAGGAGC TGCAGGTGAT TCAGCCTGAC AAGTCCGTAT CAGTTGCAGC TGGAGAGTCG GCCATTCTGC ACTGCACTGT GACCTCCCTG ATCCCTGTGG GGCCCATCCA GTGGTTCAGA GGAGCTGGAC CAGCCCGGGA ATTAATCTAC AATCAAAAAG AAGGCCACTT CCCCCGGGTA ACAACTGTTT CAGAGTCCAC AAAGAGAGAA AACATGGACT TTTCCATCAG CATCAGTGCC ATCACCCCAG CAGATGCCGG CACCTACTAC TGTGTGAAGT TCCGGAAAGG GAGCCCTGAC ACGGAGTTTA AGTCTGGAGC AGGCACTGAG CTGTCTGTGC GTGCCAAACC CTCTGCCCCC GTGGTATCGG GCCCTGCGGC GAGGGCCACA CCTCAGCACG AATTCGAGCC CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTTG A 1101 SIRP alpha Dl-Fc SEQ ID NO: 14 EEELQVIQPD KSVSVAAGES AILHCTVTSL IPVGPIQWFR GAGPARELIY NQKEGHFPRV TTVSESTKRE NMDFSISISA ITPADAGTYY CVKFRKGSPD TEFKSGAGTE LSVRAKPSAP VVSGPAARAT PQHEFEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 366 SIRP alpha Dl-Linker-anti-EGFR light chain SEQ ID NO: 15 GAGGAGGAGC TGCAGGTGAT TCAGCCTGAC AAGTCCGTAT CAGTTGCAGC TGGAGAGTCG GCCATTCTGC ACTGCACTGT GACCTCCCTG ATCCCTGTGG GGCCCATCCA GTGGTTCAGA GGAGCTGGAC CAGCCCGGGA ATTAATCTAC AATCAAAAAG AAGGCCACTT CCCCCGGGTA ACAACTGTTT CAGAGTCCAC AAAGAGAGAA AACATGGACT TTTCCATCAG CATCAGTGCC ATCACCCCAG CAGATGCCGG CACCTACTAC TGTGTGAAGT TCCGGAAAGG GAGCCCTGAC ACGGAGTTTA AGTCTGGAGC AGGCACTGAG CTGTCTGTGC GTGCCAAACC CTCTGGCGGC GGTGGGAGCG GCGGCGGTGG GAGCGGCGGC GGGGGCTCGC AGGTGCAGCT GAAGCAGTCA GGACCTGGCC TAGTGCAGCC CTCACAGAGC CTGTCCATCA CCTGCACAGT CTCTGGTTTC TCATTAACTA ACTATGGTGT ACACTGGGTT CGCCAGTCTC CAGGAAAGGG TCTGGAGTGG CTGGGAGTGA TATGGAGTGG TGGAAACACA GACTATAATA CACCTTTCAC ATCCAGACTG AGCATCAACA AGGACAATTC CAAGAGCCAA GTTTTCTTTA AAATGAACAG TCTGCAATCT CAGGACACAG CCATATATTA CTGTGCCAGA GCCCTCACCT ACTATGATTA CGAGTTTGCT TACTGGGGCC AAGGGACTCT GGTCACTGTC TCTGCAGCTA GCACCAAGGG CCCATCGGTC TTCCCCCTGG CACCCTCCTC CAAGAGCACC TCTGGGGGCA CAGCGGCCCT GGGCTGCCTG GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA ACTCAGGCGC CCTGACCAGC GGCGTGCACA CCTTCCCGGC TGTCCTACAG TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAG CTTGGGCACC CAGACCTACA TCTGCAACGT GAATCACAAG CCCAGCAACA CCAAGGTGGA CAAGAGAGTT GAGCCCAAAT CTTGTGACAA AACTCACACA TGCCCACCGT GCCCAGCACC TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTATG TGGACGGCGT GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACGCCA CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAAGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT CGCCGCAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT ACACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA AGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTATTCCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGCAAATGA 1749 SIRP alpha D1-Linker-anti-EGFR light chain SEQ ID NO: 16 EEELQVIQPD KSVSVAAGES AILHCTVTSL IPVGPIQWFR GAGPARELIY NQKEGHFPRV TTVSESTKRE NMDFSISISA ITPADAGTYY CVKFRKGSPD TEFKSGAGTE LSVRAKPSGG GGSGGGGSGG GGSQVQLKQS GPGLVQPSQS LSITCTVSGF SLTNYGVHWV RQSPGKGLEW LGVIWSGGNT DYNTPFTSRL SINKDNSKSQ VFFKMNSLQS QDTAIYYCAR ALTYYDYEFA YWGQGTLVTV SAASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNATYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIAAT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 582 Light chain of anti-EGFR antibody SEQ ID NO: 17 GACATCTTGC TGACTCAGTC TCCAGTCATC CTGTCTGTGA GTCCAGGAGA AAGAGTCAGT TTCTCCTGCA GGGCCAGTCA GAGTATTGGC ACAAACATAC ACTGGTATCA GCAAAGAACA AATGGTTCTC CAAGGCTTCT CATAAAGTAT GCTTCTGAGT CTATCTCTGG GATCCCTTCC AGGTTTAGTG GCAGTGGATC AGGGACAGAT TTTACTCTTA GCATCAACAG TGTGGAGTCT GAAGATATTG CAGATTATTA CTGTCAACAA AATAATAACT GGCCAACCAC GTTCGGTGCT GGGACCAAGC TGGAGCTGAA ACGAACTGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG 645 Light chain of anti-EGFR antibody SEQ ID NO: 18 DILLTQSPVI LSVSPGERVS FSCRASQSIG TNIHWYQQRT NGSPRLLIKY ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYCQQ NNNWPTTFGA GTKLELKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 signal peptide of mouse IgGl heavy chain SEQ ID NO: 19 ATGGGATGGT CATGTATCAT CCTTTTTCTG GTAGCAACTG CAACTGGAGT ACATTCA 57 SEQ ID NO: 20 GCCGCCACC Linker SEQ ID NO: 21 GGGGS GGGGS GGGGS 15
[0153] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.