western range biopharmaceuticals pvt ltd - carerm · western range biopharmaceuticals pvt ltd...

Western Range Biopharmaceuticals Pvt Ltd

Western Range Biopharmaceuticals Pvt Ltd (CIN U24119MH2012PTC226409) Registered Office: E-5, Kasliwal Vishwa, Parvati Nagar, Ulkanagari, Aurangabad – 431005

Branch Office: 201, Sarthik Square, Near GNFC Infotower, S.G. Highway, Ahmedabad – 380054

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Standard Operating Procedure

Thera-P-nk


Version 1.0

Dr Vinod Kuberkar Dr Senthilkumar Natesan M.Tech, PhD, MBA MVSc, PhD CEO Chief Scientific Officer




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Immunotherapy Immunotherapy, whichworks by manipulating patients own immune system to fight cancer, is a rapidly advancing field of translational science. It has been recognized as ‘breakthrough of the year 2013’ by the journal Science. Cellular immunotherapy takes advantage of activated immune cells including dendritic cells, Natural killer cells, T-cells, etc. for treatment of cancers. Dr William Coley first intentionally tried to use immune system to fight cancer in 1891 when he injected bacterial toxins in a cancer patient. Over the next hundred years, there have been many case reports of spontaneous regression of patients’ cancers, which were attributed to destruction of cancer cells by the immune system. Thus, it appears that the immune system is an intricate variable in the prevention (and possibly even in the formation) of malignant neoplasm. The strategies utilized by the immune system to fight cancer are largely the same methods used to fight infectious diseases. The difference lies in the fact that tumor antigens are human proteins that have been adapted to participate in carcinogenesis while pathogenic antigens are transcribed from their own, foreign genomes. Over the last two decades, a large number of academic institutions and companies have started pursuing cellular immunotherapy approach for treatment of cancer. NK cell therapy Natural Killer (NK) cells were first described in 1975 as a distinct subset of lymphocytes, which are larger in size than T and B lymphocytes, and contain distinctive cytoplasmic granules (Kiessling et al, 1975 and Herberman et al, 1975). They also showed profound tumor cell killing property without any need for prior immune sensitization of the host or antigen presentation. In humans, NK cells are characterized by expression of surface markers CD56 (NCAM-1) and CD16 (FcRIII). Functionally, NK cells are capable of killing as well as inducing tolerance depending on their activation state. In normal steady state, they express both activating and inhibitory receptors, where the resulting effect depends on the balance of engaged activating and inhibitory receptors. NK cells play a critical role in cellular host immunity against cancer, and the cancer cells also develop mechanisms to escape NK cell mediated immunity. Currently, the most common NK cell-based cancer immunotherapy approach is based on the adoptive transfer ofexpanded autologous NK cells. However, stable allogeneic NK cells, genetically modified NK cells, etc have also been used to a limited extent.Adoptively transferred ex vivo expanded activated autologous NK cells have been shown to improve clinical responses without any obvious adverse side effects in patients against different cancers including metastatic renal cell carcinoma (RCC), malignant glioma, and breast cancer. NK cells have the ability to recognize the cancer cells and kill them directly. They can also recognize antibody-coated target cells and trigger NK cell-mediated immune response resulting in rapid killing of cancer cells.




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Pictorial Depiction of the function of NK cells

Bright field images show very active proliferation and expansion of the NK cells in the culture

Expanded NK cells- surface marker expression Expanded NK cells- cytotoxicity Flow cytometric characterization of NK cells for their surface marker expression. CD56 is the marker for NK cells.Increase in the ratio of cancer cell to effector NK cells showed more efficient killing of cancer cells in the cytotoxicity assay

In vitro cytotoxicity assay by co-culturing tumor cells with the NK cells generated from a terminally ill cancer patient’s blood in our laboratory in the 3D spheroid culture form at two ratios. In the presence of NK cells, the tumor spheroids are surrounded by the NK cells, which cause smaller and necrotizing spheroids. Images were captured on day 6 of co- culture. M-Tumor cells and T- NK cells




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Thera-P-nk: NK Cell Therapy Protocol Thera-P-nk is a cellular immunotherapy for the treatment of cancers using expanded and activated NK cells. NK cells are efficient in recognizing the cancer cells and capable of killing them effectively. Our NK cells based approach is developed for targeted killing of cancer cells. This protocol is for generation of Thera-P-nk from 50ml of blood obtained from the cancer patients

Dosage Day 1: Thera-P-nk Immunotherapy 1 bottle Day 8: Thera-P-nk Immunotherapy 1 bottle Day 15:Thera-P-nk Immunotherapy 1 bottle Day 23:Thera-P-nk Immunotherapy 1 bottle




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Procedure and protocol Collection of blood We will be providing a blood collection kit containing all the necessary items for collectionof the blood, including the collection bag with anti-coagulant, needles, swabs, etc. Blood needs to be collected in this bag and the kit box can be used for transport of the blood. Transportation of the blood The collected blood can be transported in the given box at room temperature and it should reach our processing facility within 24 hours. Upon receiving the blood, we will check the integrity of the bag and identify the sample as per the identifier in the bag and the blood will be processed immediately. Processing of the Blood The blood will be removed from the bag under aseptic conditions in the biosafety cabinet and transferred in to centrifuge tubes. The mononuclear cells will be isolated from the rest of the blood by gradient centrifugation. These mononuclear cells will be counted and the total count will be obtained. Preparation of Thera-P-nk doses We will culture the NK cells following our protocol. It will take about2-3 weeks for generation of Thera-P-nk doses. At the end of culture, from the 50ml of blood, we would be able to supply four doses of Thera-P-nk, with each dose containing approximately0.5 to 1 billion NK cells.We will provide these cells in ready-to-use form in a vial which can be mixed diluent and directly injected in to 100 ml normal saline bottle / bag just before use, and administered to the patients through the IV route using a blood transfusion IV set. Fresh cells will be given for each dose and itwill be transported in ice pack. Quality control Quality of NK cells will be assessed by their morphology, dynamic migration, cytokine secretion, and surface marker expression. As a routine quality control approach, we test all our final preparation of cell doses for any microbial contamination. Prior to preparing the cell doses, a small aliquot of the media from each culture will be taken and DNA will be prepared and then tested for presence of any bacteria, mycoplasma, and fungus by polymerase chain reaction Only the cells that pass the quality check will beused for therapeutic purposes. Preparation of cell doses, packing, and transportation WRBIO will provide the cells in ready-to-use form in 10ml vial which can be mixed with 5ml of diluent and directly injected in to 100 ml normal saline bottle just before use, and administered to the patients through the IV route using a blood transfusion IV set. Cells of each dose will be in packed in each glass vial in 4.5 ml volume and transported in cool condition. It will be transported at 4C in a gel pack from lab to destination point. Upon receiving the cells, it should be injected in to the patient within 12 hours of receipt.




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Infusion of the Thera-P-nkin the Patients For infusing the Thera-P-nk in to a patient, the following procedure should be followed strictly.

1. Take out 5ml of the diluent using a 10ml syringe fitted with 18G needle and inject it into the Thera-P-nkvial

2. Mix the content four times by sucking (very gently) the cell suspension in to the syringe and pushing it back (very gently).

3. Collect the mixed dose with diluent into the 10ml syringe and inject into a 100ml normal saline infusion bag/ bottle

4. Take 5ml of normal saline out from the saline bag and infuse into the vial and collect (very gently) the remaining cells inside the vial and inject into the saline bag

5. Mix the content of saline bag/bottle well by inverting it gently 6. Fit the bag/bottle with a blood transfusion set and infuse into the patient intravenously.

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