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CN114712494B - PD-1-targeting multifunctional antibody composition - Google Patents

PD-1-targeting multifunctional antibody composition Download PDF

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CN114712494B
CN114712494B CN202110010966.3A CN202110010966A CN114712494B CN 114712494 B CN114712494 B CN 114712494B CN 202110010966 A CN202110010966 A CN 202110010966A CN 114712494 B CN114712494 B CN 114712494B
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heavy chain
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pharmaceutical composition
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CN114712494A (en
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姜晓玲
吴崇兵
朱彩林
杜雾晨
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Sunho China Biopharmaceutical Co Ltd
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Abstract

The invention relates to a stable pharmaceutical composition of a multifunctional antibody and application thereof. In particular, the invention relates to a pharmaceutical composition comprising a multifunctional antibody targeting PD-1 while having the biological effect of an IL-15/IL-15 ra complex, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a surfactant, a sugar, and an amino acid. The pharmaceutical composition provided by the invention has higher stability and PD-1 binding force.

Description

PD-1-targeting multifunctional antibody composition
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a pharmaceutical composition of a multifunctional antibody targeting PD-1 and having biological effects of an IL-15/IL-15Rα complex.
Background
PD-1 (programmed death protein-1) is an inhibitory co-stimulatory molecule encoded by the PDCD1 gene, a 50-55kDa type I transmembrane glycoprotein consisting of 288 amino acid residues, consisting of an extracellular IgV domain, a transmembrane domain and an intracellular tail structure. It can be expressed on the surface of activated T cells, B cells, NK cells, and other monocytes and Dendritic Cells (DCs), suggesting that PD-1 plays a more critical role in immunomodulation. PD-1 as a negative co-stimulatory molecule expressed on activated T cells binds to PD-L1 expressed by tumor cells, inhibiting T cell proliferation and promoting apoptosis of activated T cells; the PD-1 antibody can block the combination of PD-1/PD-L1, so that the effector T cells exert the tumor killing effect.
Cytokines are a class of signaling proteins and glycoproteins that are widely used in cellular communication, such as hormones and neurotransmitters. Although hormones are secreted into the blood by specific organs and neurotransmitters are involved in neural activity, cytokines are a more diverse class of compounds in terms of their origin and use. They are produced by a variety of hematopoietic and non-hematopoietic cell types and can have an effect on neighboring cells or whole organisms, sometimes strongly dependent on the presence of other chemicals. The cytokine family consists mainly of smaller water-soluble proteins and glycoproteins with masses between 8 and 30 kDa. Cytokines are important for the function of both the innate immune response and the adaptive immune response. Are normally secreted by immune cells that have encountered pathogens as a way to activate and recruit more immune cells and increase the system response to the pathogen.
Among cytokines, interleukin 15 (IL-15) is a member of the IL-2 superfamily that is secreted by many cell types and tissues, including monocytes, macrophages, dendritic Cells (DCs), keratinocytes, fibroblasts, and nerve cells. IL15 is listed by the united states national cancer center as the most potential cancer immunotherapeutic agent, with the name 20 most potential tumor immunotherapeutic targets and 12 most promising tumor cure targets first. Studies have shown that IL-15 can effectively activate proliferation and activation of CD8+ T cells, NK cells and NK cells in immune lines, and these three types of cells are the most important immune cells in tumor immunity.
However, the clinical use of cytokines has the disadvantage that the targeting of single administration is poor, and only high concentrations of administration can achieve the antitumor effect, while high concentrations of administration can produce immunosuppression and high toxicity. Moreover, non-targeted cytokines are systemic for the activation of the immune system, which is widely activated with fatal side effects. In addition, because cytokines belong to small molecular weight proteins, and do not have an in vivo circulation protection mechanism of antibodies, simple cytokines often have a short half-life and need to be repeatedly administered in high doses in a short time. At present, the clinical research drugs mostly adopt PEGylation or Fc fusion to improve the half-life of the cytokines, but the problem of poor targeting of the cytokines cannot be solved although the half-life is prolonged.
Both IL15R alpha and IL-15 are expressed in DC cells and monocytes, and the primary role of IL15R alpha is to, after binding to IL-15, present the bound IL-15 to IL2R beta and IL2R gamma, thereby stimulating proliferation and activation of immune cells, whereby the development of IL-15 activity is dependent on the presentation of IL-15R alpha. However, the expression level of DC cells IL-15Ra in resting state is very low, and the treatment of IL-15 alone administered to patients has limited clinical efficacy, so that the formation of IL15/IL15Rα and IL15Rα IgFc complexes solves the problem. Studies have shown that the biological activity of the IL15/IL15Rα complex can be increased by a factor of 100 compared with that of IL-15 alone, and fusion with FC effectively prolongs the half-life of the antibody.
In the prior art, CN110214147A and CN110214148A both disclose fusion proteins containing IL-15/IL-15Ra, solving the problem that short half-life prevents advantageous drugs. However, the disclosed antibody comprising the IL-15/IL-15Ra structure still has the problem of light and heavy chain mismatch due to its complex structure, and there is no study on the related preparation with high stability.
The invention creatively embeds IL-15 and its receptor fragment in the antibody molecular chain, designs the two to be mutually combined, and uses the special combining action of the two to facilitate the hetero dimerization of the antibody, thus the light and heavy chain mismatch problem common to the common bispecific antibody can not occur. Meanwhile, through research on the preparation of the multifunctional antibody, a multifunctional antibody composition with higher stability and better affinity with the PD-1 end is developed, the quality control is easier, and the production process and the patentability are more stable.
Disclosure of Invention
In order to solve the problems in the prior art, the invention discloses a multifunctional antibody composition which has higher affinity, long half-life, good preparation stability and lower production cost of drug molecules.
The pharmaceutical composition of the multifunctional antibody comprises the multifunctional antibody and a buffer; the buffer is selected from one or more of citric acid-sodium citrate, acetic acid-sodium acetate and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; the pH of the buffer is 5.5-6.5, preferably 6.0.
In an alternative embodiment, the buffer is present at a concentration of 1 to 100mM, preferably 5 to 50mM, more preferably 20mM.
In an alternative embodiment, the concentration of the multifunctional antibody is 0.5-50mg/ml, preferably 1-20mg/ml, more preferably 2-10mg/ml, most preferably 5mg/ml.
In an alternative embodiment, the pharmaceutical composition further comprises a surfactant, preferably the surfactant is selected from polysorbate-80 or polysorbate-20, more preferably the surfactant is polysorbate-80.
In alternative embodiments, the concentration of the surfactant is from 0.01 to 1mg/ml, preferably from 0.05 to 0.8mg/ml, more preferably from 0.2 to 0.6mg/ml, most preferably 0.2mg/ml or 0.4mg/ml.
In an alternative embodiment, the pharmaceutical composition further comprises an alcohol, preferably selected from ethanol, mannitol or sorbitol.
In an alternative embodiment, the alcohol concentration is 0-300mM, preferably 10-200mM, more preferably 20-100mM.
In an alternative embodiment, the pharmaceutical composition further comprises a sugar, preferably selected from trehalose, sucrose or dextran-40.
In an alternative embodiment, the concentration of the sugar is 0-200mM, preferably 5-100mM, more preferably 10-80mM, most preferably 20-50mM.
In alternative embodiments, the multifunctional antibody comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain, a portion of the first heavy chain and a portion of the first light chain, a portion of the second heavy chain and a portion of the second light chain are paired, respectively, and one or both form a PD-1 antigen binding site, the first heavy chain further comprising a cytokine IL-15 fragment and an immunoglobulin Fc portion, the second heavy chain further comprising an IL-15 receptor fragment and an immunoglobulin Fc portion, the cytokine IL-15 fragment in the first heavy chain and the IL-15 receptor fragment in the second heavy chain being mutually bound.
In alternative embodiments, the immunoglobulin Fc portion of the first and second heavy chains is selected from the group consisting of constant region amino acid sequences of IgG1, igG2, igG3 and/or IgG4, preferably constant region amino acid sequences of IgG1 or IgG 4.
In alternative embodiments, the IL-15 fragment in the first heavy chain and the IL-15 receptor fragment in the second heavy chain may each be chimeric within the Fc portion of the chain, or may be present outside the Fc portion, preferably between the CH1 and CH2 functional regions of the respective heavy chains.
In an alternative embodiment, the first heavy chain amino acid sequence of the multifunctional antibody is selected from the group consisting of SEQ ID NO. 1; the second heavy chain amino acid sequence of the multifunctional antibody is selected from SEQ ID NO. 3; the amino acid sequences of the first light chain and the second light chain of the multifunctional antibody are selected from SEQ ID NO. 5.
In an alternative embodiment, a lyophilized formulation comprising a multifunctional antibody is obtained by lyophilizing the pharmaceutical composition described above.
In an alternative embodiment, a liquid formulation comprising a multifunctional antibody is obtained by aseptically filling the pharmaceutical composition described above.
In an alternative embodiment, the liquid formulation is an injectable formulation, preferably the injectable formulation is a water needle.
In an alternative embodiment, the use of the above pharmaceutical composition or lyophilized formulation or liquid formulation for the manufacture of a medicament for the prevention or treatment of a disease or disorder associated with a PD-1 antigen, such as a tumor.
Accordingly, the present invention relates to the following embodiments:
1. a pharmaceutical composition of a multifunctional antibody, wherein the pharmaceutical composition comprises a multifunctional antibody and a buffer; the buffer is selected from one or more of citric acid-sodium citrate, acetic acid-sodium acetate and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; the pH of the buffer is 5.5-6.5.
2. The pharmaceutical composition according to scheme 1, characterized in that the concentration of the buffer is 1-100mM, preferably 5-50mM.
3. The pharmaceutical composition according to scheme 1 or 2, characterized in that the concentration of the multifunctional antibody is 0.5-50mg/ml, preferably 1-20mg/ml, more preferably 2-10mg/ml.
4. A pharmaceutical composition according to any one of the claims 1-3, characterized in that the pharmaceutical composition further comprises a surfactant, preferably the surfactant is selected from polysorbate-80 or polysorbate-20, more preferably the surfactant is polysorbate-80.
5. The pharmaceutical composition according to scheme 4, characterized in that the concentration of the surfactant is 0.01-1mg/ml, preferably 0.05-0.8mg/ml, more preferably 0.2-0.6mg/ml.
6. The pharmaceutical composition according to any one of the claims 1-5, wherein the pharmaceutical composition further comprises an alcohol, preferably the alcohol is selected from ethanol, mannitol or sorbitol.
7. The pharmaceutical composition according to scheme 6, characterized in that the concentration of the alcohol is 0-300mM, preferably 10-200mM, more preferably 20-100mM.
8. The pharmaceutical composition according to any one of the claims 1-7, characterized in that the pharmaceutical composition further comprises a sugar, preferably the sugar is selected from trehalose, sucrose or dextran-40.
9. The pharmaceutical composition according to claim 8, wherein the concentration of the sugar is 0-200mM, preferably 5-100mM, more preferably 10-80mM.
10. The pharmaceutical composition of any one of claims 1-9, wherein the multifunctional antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein a portion of the first heavy chain and a portion of the first light chain, a portion of the second heavy chain, and a portion of the second light chain are paired, respectively, and one or both form a PD-1 antigen-binding site, wherein the first heavy chain further comprises a cytokine IL-15 fragment and an immunoglobulin Fc portion, wherein the second heavy chain further comprises an IL-15 receptor fragment and an immunoglobulin Fc portion, and wherein the cytokine IL-15 fragment in the first heavy chain and the IL-15 receptor fragment in the second heavy chain are mutually bound.
11. The pharmaceutical composition according to claim 10, wherein the immunoglobulin Fc portion of the first and second heavy chains is selected from the group consisting of constant region amino acid sequences of IgG1, igG2, igG3 and/or IgG4, preferably constant region amino acid sequences of IgG1 or IgG 4.
12. The pharmaceutical composition according to claim 10 or 11, wherein the IL-15 fragment in the first heavy chain and the IL-15 receptor fragment in the second heavy chain may be chimeric within the Fc portion of the chain, respectively, or may be present outside the Fc portion, preferably between the CH1 and CH2 functional regions of the respective heavy chains.
13. The pharmaceutical composition of any one of claims 1-12, wherein the first heavy chain amino acid sequence of the multifunctional antibody is selected from the group consisting of SEQ ID No. 1; the second heavy chain amino acid sequence of the multifunctional antibody is selected from SEQ ID NO. 3; the amino acid sequences of the first light chain and the second light chain of the multifunctional antibody are selected from SEQ ID NO. 5.
14. A lyophilized formulation comprising a multifunctional antibody, wherein the formulation is obtained by lyophilizing the pharmaceutical composition of any one of schemes 1 to 13.
15. A liquid formulation comprising a multifunctional antibody, characterized in that the formulation is obtained by aseptically filling the pharmaceutical composition according to any one of schemes 1 to 13.
16. The liquid formulation according to claim 15, wherein the liquid formulation is an injection formulation, preferably the injection formulation is a water needle.
17. Use of a pharmaceutical composition according to any one of schemes 1-13 or a lyophilized formulation according to scheme 14 or a liquid formulation according to scheme 15 or 16 for the manufacture of a medicament for the prevention or treatment of a disease or disorder associated with a PD-1 antigen, such as a tumor.
Advantageous effects
The invention provides an antibody pharmaceutical composition with better pharmacodynamics, higher purity and more stability, which creatively embeds IL-15 and a receptor fragment thereof in an antibody molecular chain, designs the IL-15 and the receptor fragment thereof as mutually-combinable positions, and utilizes the special combination effect of the IL-15 and the receptor fragment to facilitate the heterodimerization of the antibody, so that the problem of light and heavy chain mismatch common to common bispecific antibodies does not occur.
Furthermore, the multifunctional antibody obtained by the invention has high-efficiency PD-1 antigen affinity, and the composition of the invention has good stability, is not easy to generate polymer and small molecule fragments, and still maintains higher antibody purity after long-time storage.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Terminology
"Buffering agent" refers to a buffering agent that is resistant to pH changes by the action of its acid-base conjugate components. Examples of buffers to control the pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.
A "phosphate buffer" is a buffer that includes phosphate ions, including disodium hydrogen phosphate/sodium dihydrogen phosphate.
An "acetate buffer" is the buffer histidine/acetic acid that includes acetate ions.
"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredients of the antibody, promote the administration to organisms, and facilitate the absorption of the active ingredients so as to exert biological activity.
As used herein, the terms "pharmaceutical composition" and "formulation" are not intended to be mutually exclusive.
As used herein, the terms "buffer" and "buffer system" are not mutually exclusive.
The pharmaceutical composition of the present invention is in the form of a solution, wherein the solvent is water unless otherwise specified.
By "lyophilized formulation" is meant a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a liquid or solution formulation.
Drawings
FIG. 1a is a plasmid pcDNA3.1-G418-16-1 containing the first heavy chain of a multifunctional antibody;
FIG. 1b is a plasmid pcDNA3.1-G418-16-2 containing a second heavy chain of a multifunctional antibody;
FIG. 1c is a plasmid pcDNA3.1-G418-16-3 containing the first/second light chain (both identical) of a multifunctional antibody.
FIG. 2a is a SDS-PAGE under non-reducing conditions;
FIG. 2b is a SDS-PAGE under reducing conditions.
FIGS. 3a to 3c are schematic diagrams of ELISA results for binding of multifunctional antibodies to PD-1 antigen.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, to which the invention is not limited. It is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Variations and advantages that will occur to those skilled in the art are included within the following claims and any equivalents thereof without departing from the spirit and scope of the inventive concept. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge to those skilled in the art, except where specifically mentioned below, and the present invention is not particularly limited.
Example 1 nucleotide sequence acquisition and optimization
The amino acid sequence information of the light chain and the heavy chain of the multifunctional antibody is selected from published or self-developed PD-1 target monoclonal antibody sequence information, and the variable region and the constant region information of the sequence are obtained by analysis. The native IL-15 sequence or IL-15 variant sequence is inserted into the amino acid sequence of one heavy chain and an IL-15 receptor sequence, preferably an IL-15Rα Sushi sequence, is inserted at the corresponding position of the other heavy chain. The Fc of the amino acid sequence of the antibody is adjusted to other IgG types, such as IgG1, etc., and further amino acid mutations of a desired form are designed in each heavy chain, thereby obtaining the amino acid sequence of the target antibody, which is respectively:
The first heavy chain is SEQ ID NO. 1, the second heavy chain is SEQ ID NO. 3, and the first light chain and the second light chain (both identical) are SEQ ID NO. 5.
Converting each of the above-described amino acid sequences of interest into a nucleotide sequence, and targeting a series of parameters that may affect the expression of the antibody in mammalian cells: codon preference, GC content (i.e. the ratio of guanine G and cytosine C in 4 bases of DNA), cpG islands (i.e. the region of higher density of CpG dinucleotides in the genome), secondary structure of mRNA, splice sites, pre-mature PolyA sites, internal Chi sites (a short DNA fragment in the genome, increased probability of homologous recombination occurring near this site) or ribosome binding sites, RNA instability sequences, inverted repeats, restriction sites that might interfere with cloning, etc.; related sequences, such as Kozak sequences, SD sequences, and stop codons, which may increase translation efficiency, are also added. Designing a heavy chain gene and a light chain gene for encoding the multifunctional antibody molecule, and designing nucleotide sequences for encoding signal peptides obtained by optimizing amino acid sequences at the 5' ends of the heavy chain and the light chain respectively; in addition, stop codons were added to the 3' ends of the light and heavy chain nucleotide sequences, respectively.
Finally optimizing to obtain the multifunctional antibody optimized nucleotide sequence, which is:
The first heavy chain is SEQ ID NO. 2, the second heavy chain is SEQ ID NO. 4, and the first light chain and the second light chain (both identical) are SEQ ID NO. 6.
EXAMPLE 2 Gene synthesis and construction of expression vectors
The pcDNA3.1-G418 vector is adopted as a special vector for expressing the light chain and the heavy chain of the multifunctional antibody. The pcDNA3.1-G418 vector contains a promoter CMVPromoter for the heavy chain, a eukaryotic selectable marker G418 tag and a prokaryotic selectable tag AMPICILLINE. The nucleotide sequences of the antibody expression light chain and heavy chain of the multifunctional antibody are obtained by gene synthesis, hindIII and XhoI are used for carrying out double enzyme digestion on the vector and the target fragment, the vector and the target fragment are subjected to enzyme ligation through DNA ligase after being recovered, and E.coli competent cells DH5 alpha are transformed, positive clones are selected, plasmid extraction and enzyme digestion verification are carried out, and recombinant plasmids containing the full-length first heavy chain, the second heavy chain, the first light chain and the second light chain of the multifunctional antibody are obtained, wherein the recombinant plasmids are pcDNA3.1-G418-16-1, pcDNA3.1-G418-16-2 and pcDNA3.1-G418-16-3 (the first light chain and the second light chain are identical). Exemplary plasmid maps of pcDNA3.1-G418-16-1, pcDNA3.1-G418-16-2 and pcDNA3.1-G418-16-3 are shown in FIGS. 1a-1c.
EXAMPLE 3 plasmid extraction
The recombinant plasmids containing the above-mentioned genes were transformed into competent cells DH 5. Alpha. Of E.coli according to the method described in the guidelines for molecular cloning experiments (2002, scientific Press), the transformed bacteria were spread on LB plates containing 100. Mu.g/ml ampicillin, the plasmid clones were selected and cultured in liquid LB medium, shaking at 260rpm for 14 hours, the plasmids were extracted by endotoxin-free plasmid megapump kit, dissolved in sterile water and assayed for concentration using a nucleic acid protein quantitative analyzer.
EXAMPLE 4 plasmid transfection, transient expression and antibody purification
ExpiCHO was cultured at 37℃and 8% CO 2 at 100rpm to a cell density of 6X 106cells/ml. The constructed vectors PCDNA 3.1.1-G418-16-1, PCDNA 3.1.1-G418-16-2, PCDNA 3.1.1-G418-16-3 were transfected into the above cells using liposomes at a concentration of 1. Mu.g/ml, respectively, as determined by reference to ExpiCHO TM expression System kit, and cultured at 32℃for 7-10 days at 5% CO 2, 100 rpm. Feed was fed once between 18-22h and 5-8 days after transfection, respectively. The above culture product was centrifuged at 4000rpm, filtered through a 0.22 μm filter membrane and the culture supernatant was collected, and the resulting multifunctional antibody protein was purified by using a protease A and an ion column and the eluate was collected.
The specific operation steps of ProteinA and ion column purification are as follows: the cell culture fluid is centrifuged at high speed, and the supernatant is subjected to affinity chromatography by using a GE protein A chromatography column. Chromatography uses equilibration buffer 1 XPBS (pH 7.4), cell supernatants were combined, washed with PBS to UV light back to baseline, then eluted with elution buffer 0.1M glycine (pH 3.0), and stored with Tris to adjust pH to neutral. The pH of the product obtained by affinity chromatography is adjusted to a pH unit of 1-2 below or above pI, and the product is diluted appropriately to control the sample conductance below 5 ms/cm. And (3) performing NaCl gradient elution under the corresponding pH conditions by utilizing proper corresponding pH buffers such as phosphate buffer, acetate buffer and the like and utilizing ion exchange chromatography methods such as anion exchange or cation exchange which are conventional in the field, and selecting a collecting tube in which the target protein is positioned according to SDS-PAGE, and combining and storing.
SDS-PAGE determination shows that under non-reducing condition, the molecular weight of the expressed complete multifunctional antibody is slightly higher than that of the IgG1 antibody; igG1 antibodies were reduced to 2 bands under reducing conditions, while multifunctional antibodies were reduced to 3 bands, i.e. the proteins of interest at about 64kDa, 58kDa and 24kDa, corresponding to two different heavy chains and the same light chain of the desired antibodies. Therefore, the plasmid transfection, transient expression and purification prove that the obtained antibody has correct structure and higher purity. The corresponding SDS-PAGE patterns of the multifunctional antibodies are shown in FIGS. 2a and 2 b.
Example 5 screening of antibody preparation buffer System
And (3) carrying out ultrafiltration liquid exchange on the antibody to different buffer solutions, respectively adding sucrose and polysorbate-80 after the buffer solution is replaced, maintaining the final concentration to be 8% (w/v) of sucrose and 0.02% (w/v) of polysorbate-80, sterilizing, filtering, and carrying out sterile packaging.
The different buffer system protocols are shown in table 1.
Table 1 buffer System protocol for antibodies
The prepared antibody preparation was subjected to detection by size exclusion high performance liquid chromatography (SEC-HPLC) at 25deg.C and at week 3 and week 4, respectively, and the purities of the antibody preparation were used for 3 weeks (3W) and 4 weeks (4W), and the influence of different buffers on the stability of the preparation was examined, and the experimental results are shown in Table 2
TABLE 2 screening test results for different buffers for antibodies
Note that: HMW represents high molecular weight; LMW means low molecular weight.
The results show that when citric acid-sodium citrate, acetic acid-sodium acetate and histidine-histidine hydrochloride are used as the buffering agent, the main peak purity is lower at week 3 and week 4, and the standards are not met. When citric acid-sodium citrate and acetic acid-sodium acetate were used as the buffer, the high and low molecular weight contents were relatively high at weeks 3 and 4, indicating that the antibody preparation produced more polymer and small molecular fragments and that the antibody preparation had poor stability. When histidine-histidine hydrochloride was used as the buffer, the high molecular weight was relatively low at weeks 3 and 4, but the low molecular weight was very high, indicating that the antibody preparation produced a large number of small molecule fragments and that the antibody preparation was not stable. When disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is used as the buffer, the main peak purity is relatively high at week 3 and week 4, and the contents of high molecular weight and low molecular weight are relatively low, which means that the antibody purity is relatively high, the stability is good, and the generation of polymers and small molecular fragments is not easy.
Example 6 surfactant screening
Using the different surfactants shown in Table 3, 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and 135mM trehalose were added to prepare 5mg/ml antibody preparations. The effect of polysorbate 20 and polysorbate 80 having different contents on formulation was examined, and repeated freeze-thawing cycles (cycle temperature: -80-4 ℃) and shaking for 7 days (250 rpm,25 ℃) were performed, respectively, and the experimental results are shown in Table 4.
Table 3 surfactant protocols for antibody compositions
Table 4 surfactant protocol results for antibody compositions
Note that: HMW represents high molecular weight, DR-2 is repeated freeze thawing cycle (circulation temperature: 80-4deg.C) 2 times, DR-5 is repeated freeze thawing cycle (circulation temperature: 80-4deg.C) 5 times; ZD-7D was shaken for 7 days (250 rpm,25 ℃).
Example 7 screening of antibody preparation stabilizers
To further optimize the stabilizer species, antibody preparations containing 5mg/ml mab, 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate, pH 6.0 were prepared in formulations containing different stabilizers as shown in Table 5.
Table 5 stabilizer protocol for antibody compositions
And (3) carrying out ultrafiltration liquid exchange on the antibody to buffer solutions of different stabilizers, and carrying out sterilization filtration and sterile split charging. The antibody preparation prepared above was placed at 40℃and removed at week 2 and week 4, respectively, for detection by size exclusion high performance liquid chromatography (SEC-HPLC) and the effect of the different stabilizers on the stability of the preparation was examined with respect to the purity at the beginning of the experiment (T0), at week 2 (2W) and at week 4 (4W), and the results are shown in Table 6.
TABLE 6 results of stabilizer protocol for antibody compositions
Note that: HMW represents high molecular weight and LMW represents low molecular weight.
The results of the antibody composition at 40 ℃ for 2 weeks and 4 weeks showed that:
(1) Prescription F1 without any stabilizer added to the composition, prescription F2 with only sucrose as stabilizer in the composition, prescription F3, F4 with only arginine as stabilizer in the composition, prescription F5, F6, F7 with only glycine as stabilizer in the composition, and polymer increase significantly with time.
(2) The stabilizer in the composition comprises schemes F8, F9 of sucrose and mannitol, the stabilizer in the composition comprises only schemes F10, F11, F12 of mannitol, the stabilizer in the composition comprises only scheme F13 of sorbitol, and the increase of polymer and the decrease of main peak purity are less with the increase of time.
(3) The compositions of schemes F2, F7 and F12 were observed to precipitate.
Example 8ELISA detection of affinity of antibodies to PD-1 antigen
PD1 antigen was diluted to 0.2. Mu.g/mL with PBS buffer pH7.4, 100. Mu.L per well was added to a 96-well ELISA plate, and 4℃coated overnight. After blocking with blocking solution containing 2% BSA for 1.5 hours. After 3 PBST washes, the multifunctional antibody was diluted to 0.3 μg/ml with 0.5% bsa sample dilution, and 3-fold gradient dilution was performed with the initial concentration, 7 gradients were added, and negative control was set up at 100 μl per well and incubated for 1h at 37 ℃. The plates were washed 3 more times with PBST, and HRP-labeled goat anti-human IgGFc was diluted 1:20000 with sample dilution, 100. Mu.L per well was added and incubated at room temperature for 1 hour. After washing the plates 4 times with PBST, 100. Mu.L of LTMB substrate was added to each well, incubated at room temperature for 10 minutes in the dark, and 100. Mu.L of 1MHCL solution was added to each well to terminate the chromogenic reaction. The absorbance of each well in a 96-well plate was determined by selecting a wavelength of 450nm on a multifunctional microplate reader and a reference wavelength of 570nm, absorbance per well (OD) =od 45nm-OD570nm. Taking the logarithm of the concentration of the multifunctional antibody as an abscissa, and taking the measured absorbance value of each hole as an ordinate, the binding curve of the multifunctional antibody and the PD1 antigen is obtained, as shown in figures 3a-3 c. The EC50 values measured are shown in tables 7 and 8.
Table 7 EC50 values for samples
Sample numbering EC50
F1-T0 4.084
F1-6W 9.24
F9-6W 5.901
Table 8 EC50 values for samples
Sample numbering EC50
F1-T0 4.349
F10-6W 9.224
F12-6W 8.772
From the PD-1 end binding activity, the activities of F1, F10 and F12 decreased after 6 weeks of high temperature acceleration, and the F9 and T0 initial sample ratios were not significantly different, indicating that the affinity of F9 to PD-1 end was higher.
While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.
Sequence listing
<110> Shenghe (China) biopharmaceutical Co., ltd
<120> A composition of multifunctional antibodies targeting PD-1
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Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala
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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
225 230 235 240
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
245 250 255
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
260 265 270
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
275 280 285
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser
290 295 300
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
305 310 315 320
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
325 330 335
Met Phe Ile Asn Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
340 345 350
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
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Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
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405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
420 425 430
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450 455 460
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Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
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<210> 2
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caggtgcagc tggtgcagag cggagtggag gtgaagaagc caggcgccag cgtgaaggtg 60
tcttgcaagg cttccggcta cacattcacc aactactata tgtattgggt gagacaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ccaatggcgg caccaacttc 180
aatgagaagt ttaagaatcg cgtgacactg accacagatt ccagcaccac aaccgcttac 240
atggagctga agagcctgca gttcgacgat accgccgtgt actattgtgc taggcgggac 300
tacaggttcg atatgggctt tgactattgg ggccagggca caaccgtgac cgtgtcttcc 360
gccagcacaa agggcccatc cgtgttccca ctggctccat gctcccggag cacctctgag 420
tccacagccg ctctgggctg tctggtgaag gactatttcc ctgagccagt gacagtgtct 480
tggaactccg gcgccctgac cagcggagtg cacacatttc ccgctgtgct gcagagctct 540
ggcctgtact ctctgtccag cgtggtgacc gtgccatctt ccagcctggg cacaaagacc 600
tatacatgca acgtggatca taagcccagc aatacaaagg tggacaagag ggtgggagga 660
ggaggatccg gaggaggagg aagcaactgg gtgaatgtga tcagcgatct gaagaagatc 720
gaggacctga tccagtctat gcacatcgat gccaccctgt acacagagtc tgacgtgcat 780
ccttcctgca aggtgaccgc tatgaagtgt tttctgctgg agctgcaggt catctccctg 840
gagtctggcg atgcctctat ccacgacacc gtggagaacc tgatcatcct ggctaatgat 900
tccctgtctt ccaacggcaa tgtgacagag agcggctgca aggagtgtga ggagctggag 960
gagaagaaca tcaaggagtt cctgcagtct tttgtgcata tcgtgcagat gttcatcaat 1020
accagcggag gaggaggatc tggaggagga ggaagcgagt ctaagtacgg accaccttgc 1080
ccaccatgtc cagctcctga gtttctggga ggaccatccg tgttcctgtt tcctccaaag 1140
cctaaggata ccctgatgat ctccagaacc cccgaggtga catgcgtggt ggtggatgtg 1200
agccaggagg accctgaggt gcagttcaac tggtacgtgg acggcgtgga ggtgcacaat 1260
gctaagacaa agcccaggga ggagcagttt aactccacct accgggtggt gagcgtgctg 1320
acagtgctgc atcaggactg gctgaacggc aaggagtata agtgcaaggt gtctaataag 1380
ggcctgccta gctctatcga gaagaccatc tccaaggcta agggacagcc tcgcgagcca 1440
caggtgtaca ccctgccccc ttctcaggag gagatgacaa agaaccaggt gtccctgtgg 1500
tgtctggtga agggcttcta tcctagcgat atcgctgtgg agtgggagtc taatggccag 1560
ccagagaaca attacaagac aaccccaccc gtgctggact ccgatggcag cttctttctg 1620
tattctagac tgaccgtgga caagtcccgc tggcaggagg gcaacgtgtt ttcctgtagc 1680
gtgatgcacg aggctctgca caatcattac acacagaagt ctctgtccct gagcctgggc 1740
aag 1743
<210> 3
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala
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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
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Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
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Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr
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Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala
225 230 235 240
Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile
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Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu
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275 280 285
Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro
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Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
305 310 315 320
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
325 330 335
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
340 345 350
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
355 360 365
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
370 375 380
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
385 390 395 400
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
405 410 415
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
420 425 430
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
435 440 445
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
450 455 460
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
465 470 475 480
Asp Gly Ser Phe Phe Leu Ala Ser Arg Leu Thr Val Asp Lys Ser Arg
485 490 495
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
500 505 510
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
515 520 525
<210> 4
<211> 1581
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
caggtgcagc tggtgcagag cggagtggag gtgaagaagc caggcgccag cgtgaaggtg 60
tcttgcaagg cttccggcta cacattcacc aactactata tgtattgggt gaggcaggct 120
ccaggacagg gactggagtg gatgggaggc atcaacccct ctaatggcgg caccaacttc 180
aatgagaagt ttaagaatcg ggtgacactg accacagatt ccagcaccac aaccgcttac 240
atggagctga agtccctgca gttcgacgat accgccgtgt actattgtgc taggcgggac 300
tacaggttcg atatgggctt tgactattgg ggccagggca caaccgtgac cgtgtcttcc 360
gccagcacaa agggcccatc cgtgttccca ctggctccat gctcccggag cacctctgag 420
tccacagccg ctctgggctg tctggtgaag gactatttcc ctgagccagt gaccgtgagc 480
tggaactctg gcgccctgac cagcggagtg cacacatttc ccgctgtgct gcagagctct 540
ggcctgtact ctctgtccag cgtggtgaca gtgccatctt ccagcctggg cacaaagacc 600
tatacatgca acgtggatca caagccctcc aataccaagg tggacaagag ggtgggagga 660
ggaggatccg gaggaggcgg cagcatcaca tgtccccctc caatgagcgt ggagcatgcc 720
gatatctggg tgaagagcta ctctctgtac tccagggaga ggtacatctg caatagcggc 780
ttcaagagaa aggctggcac ctcttccctg acagagtgcg tgctgaacaa ggccaccaat 840
gtggctcatt ggacaacccc tagcctgaag tgcatcaggg gaggaggagg atccgagagc 900
aagtatggac caccttgccc accatgtcca gctcctgagt ttctgggagg accatccgtg 960
ttcctgtttc ctccaaagcc taaggatacc ctgatgatct ccagaacccc cgaggtgaca 1020
tgcgtggtgg tggatgtgag ccaggaggac cctgaggtgc agttcaactg gtacgtggac 1080
ggcgtggagg tgcacaatgc taagaccaag cccagagagg agcagtttaa ctctacctac 1140
cgcgtggtgt ccgtgctgac agtgctgcat caggactggc tgaacggcaa ggagtataag 1200
tgcaaggtgt ctaataaggg cctgcctagc tctatcgaga agaccatctc caaggctaag 1260
ggacagcctc gcgagccaca ggtgtataca ctgcccccta gccaggagga gatgaccaag 1320
aaccaggtgt ctctgacatg tctggtgaag ggcttctacc cttctgatat cgctgtggag 1380
tgggagtcca atggccagcc agagaacaat tataagacaa ccccacccgt gctggactcc 1440
gatggcagct tctttctggc cagcaggctg accgtggaca agtctcggtg gcaggagggc 1500
aacgtgtttt cttgctccgt gatgcacgag gctctgcaca atcattacac acagaagagc 1560
ctgtctctgt ccctgggcaa g 1581
<210> 5
<211> 218
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215

Claims (7)

1. A pharmaceutical composition of a multifunctional antibody, wherein the pharmaceutical composition comprises a multifunctional antibody and a buffer; the buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; the pH of the buffer is 5.5-6.5, and the concentration of the buffer is 5-50mM; the pharmaceutical composition further comprises a surfactant, wherein the surfactant is polysorbate-80, and the concentration of the surfactant is 0.2-0.6mg/ml; the pharmaceutical composition further comprises an alcohol, wherein the alcohol is mannitol, and the concentration of the alcohol is 20-100mM; the pharmaceutical composition further comprises a sugar selected from trehalose, sucrose, at a concentration of 5-100mM; the multifunctional antibody comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain, wherein a part of the first heavy chain and a part of the first light chain, a part of the second heavy chain and a part of the second light chain are respectively paired, one or all of the two forms a PD-1 antigen binding site, the first heavy chain further comprises a cytokine IL-15 fragment and an immunoglobulin Fc part, the second heavy chain further comprises an IL-15 receptor fragment and an immunoglobulin Fc part, and the cytokine IL-15 fragment in the first heavy chain and the IL-15 receptor fragment in the second heavy chain are mutually bound; the immunoglobulin Fc portion of the first and second heavy chains is selected from the group consisting of constant region amino acid sequences of IgG1 or IgG 4; the IL-15 fragment in the first heavy chain and the IL-15 receptor fragment in the second heavy chain are located between the CH1 and CH2 functional regions of the respective heavy chains; the amino acid sequence of the first heavy chain of the multifunctional antibody is selected from SEQ ID NO. 1, the amino acid sequence of the second heavy chain is selected from SEQ ID NO. 3, and the amino acid sequences of the first light chain and the second light chain are selected from SEQ ID NO. 5.
2. The pharmaceutical composition of claim 1, wherein the concentration of the multifunctional antibody is 2-10mg/ml.
3. A lyophilized formulation comprising a multifunctional antibody, characterized in that said formulation is obtained by lyophilizing the pharmaceutical composition of claim 1 or 2.
4. A liquid formulation comprising a multifunctional antibody, characterized in that said formulation is obtained by aseptically filling a pharmaceutical composition according to claim 1 or 2.
5. The liquid formulation of claim 4, wherein the liquid formulation is an injection formulation, and the injection formulation is a water needle.
6. Use of a pharmaceutical composition according to claim 1 or 2 or a lyophilized formulation according to claim 3 or a liquid formulation according to claim 4 or 5 for the manufacture of a medicament for the prevention or treatment of a disease or disorder associated with a PD-1 antigen.
7. The use of claim 6, wherein the disease or disorder associated with the PD-1 antigen is a tumor.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018209194A2 (en) * 2017-05-12 2018-11-15 Icahn School Of Medicine At Mount Sinai Newcastle disease viruses and uses thereof
CN110214147A (en) * 2016-10-14 2019-09-06 Xencor股份有限公司 IL15/IL15R α heterodimer FC- fusion protein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110214147A (en) * 2016-10-14 2019-09-06 Xencor股份有限公司 IL15/IL15R α heterodimer FC- fusion protein
CN110214148A (en) * 2016-10-14 2019-09-06 Xencor股份有限公司 Bispecific heterodimer fusion protein containing IL-15/IL-15R α Fc fusion protein and PD-1 antibody fragment
WO2018209194A2 (en) * 2017-05-12 2018-11-15 Icahn School Of Medicine At Mount Sinai Newcastle disease viruses and uses thereof

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