Tumour Immunology Strategies

• cancer vaccines refers to therapeutic immunization in patients with cancer, with the main aim of breaking tolerance toward self-tumor-associated antigens. One strategy is to pulse antigen presenting cells such as  with tumor antigens in the hope that that such DCs will induce host protective and therapeutic antitumor immunity. 

The identification of the optimal antign to target for a pparticular tumour type has long been a priority for cancer immunotherpay. Tumour antigens can generally be categorized as being tumor associated which are overexpressed such as the HER2 receptor or tumour specific such as HPV which is associated with cervical cancer. (Hu, “Toward personalized, tumour-speciic, therapeutic vaccines for cancer” 2018). 

• Introduce genes encoding MHC Ags,  agonists, costimulatory molecules or into tumor cells. The goal here again is to improve the immunogenicity and the Ag-presenting capability of tumor cells. For example, DCs can be raised from bone-marrow stem cells upon culture with cytokine combinations. During the differentiation culture they can be transfected with recombinant viral vectors encoding cytokines like IL-12, IL-7, or IL-2. If such cells are then injected intratumorally they capture antigen, migrate to draining lymph nodes and orchestrate a potent CTL response. Artificial expression of the transgene greatly enhances the antitumor immune response. 

Interestingly, most of the pulsing has been done with peptides rather then proteins (which would require DCs to process the antigen). This is because when take DCs and put them into plastic culture dishes, giving them cytokines, etc., this matures them. So most of the DCs become mature. One way to counteracti this is to do this in viov. For example, FLT3L is a way to increase DCs in vivo to increase DCs prior to taking out the blood to grow the DCs in culture. Morese et al, JCO, 2001 found that if they systemically gaive Flt3 ligand to mice, DC population increased. Then use IL-14, GM-CSF and TNFa for growing the DCs (CD34+ HPC) in vitro.

Patients given subcutaneous GM-CSF administration increase mobilization of CD80+ DCs. Studies have shown that just giving GM-CSF is therapeutic. Newer treatments include taking tumor cells from patients, growing them, transfecting them with plasmid encoding with GMCF, then irradiating them to use them as a vacine. Trials have shown this stimulated DCs leading to increased CD8+ cells at tumor sites.

Another approach is to use TLR agonists to activate local immune response. For example, the TLR7 agonist, imidazoquinolines and TLR 9 CpG are being used to activate the innate immune system.

• Direct Tumor Transfection with Co-stimulatory Molecules: Another strategy is to take the tumor and turn it into an APC. Take tumor and put in rv-B7.1 to induce T cell activation and induce tumor recognition. Researchers have found that by doing this they were able to generate gp100- and MARt-1-specific T cells found in most A2+ patients.

• Adoptively transferring T Helper Cells to initiate CTL Immunity: Infusion of CD4+ T cell clones specific for P815AB reported shown tumor destruction through a CD8 mediated destruction. Other reports have shown that such transferring induces a broad immune response.

• Cancer Specific Antibodies: About 70 humanized monoclonal antibodies (mAbs) against tumor antigens are currently in human clinical trails, and 3 mAbs, Rituximab, herceptin and Alemtuzumab have been approved by the FDA. Although their mechanisms of action have been controversial, a major antitumor mechanisms of mAbs is thought to be by antibody-dependent cellular cytotoxicity (ADCC). mABs specific for tumor cell surface antigens bind to the tumor cell through their antigen binding domains and, through the Fc region of the mAb, bind to cells such as NK cells and neutrophils that express Fc receptors. This binding event is throught to activate the FcR-expressing cell, which results in ADCC.

• T Cell Activation with Treg Depletion: Knutson et al. 2005. Treg cells are known to express CTLA-4. Prudhomme G, J Luekocyte Biol, 2004 have given mice anti-CTLA4 and vacine against gp100. However, side effect get large autoimmunity.

• CAR T-cell Immunotherapy:  See outline

• Induce apoptosis. In models utilizing animals engrafted with human tumors, treatment with nduces significant tumor-specific apoptosis, tumor regression, and improved survival with no identifiable toxicity. TNF, LT-alpha1Beta2, FasL, and TRAIL are all expressed and used to kill cancer cells by professioanl cytotoxic cells, such as CD8+ CTLs and NK cells. In addition, Fasl, TNF, and TRAIL are expressed by activated CD4+ T cells, B cells and macrophages. Human immature DCs also express these TNF family ligands on their cell membrane. Cell surface expression and secretion of these cytotoxic ligands appears to be tightly regulated and differentiation stage-dpependent with the dendritic cell lineage. The possible regualtory mechanism might include activiteis of cytokines and/or metalloproteases.

• Induce phagocytosis. Cells undergoing apoptosis express specific signals, including lipids like phosphatidylserine (PS) that facilitate recognition and ingestion by macrophages. The possibility exists that one might be able to stimulate phagocytosis of tumor cells in the absence of a death signal to thereby delete cancer cells without even having any associated bystander effects observed during conventional chemotherapeutic treatment which is based on the induction of apoptosis.

• Attracting DCs to tumors with Chemokine: For example, injection of the chemokine CCL20 attracts DCs into the tumor, leading to induction of anti-tumor response. Intratumoral CpG can also activate tumor DC, leading to induciton of systemic anti-tumor immunity.
• Combination therapies: Strategies such as taregting multiple neopeitopes adncomining a personalized vaccines with complementary therapies will be important to prevent immune espacpe. For example, a mouse study showed that the comibnation of four components, a tumour antigen-targeting antibody, IL-2 aanti-PD1 therapy and a T cell vaccine) could liminate large tumour burdens. (Hu, “Toward personalized, tumour-speciic, therapeutic vaccines for cancer” 2018).

MedlinePlus

Most, if not all, chemotherapeutic agents kill cancer cells through the induction fo apoptosis.

Specific Chemotherapeutic agents

Colchicine: has been investigated as an anti-cancer agent and has proven to be effective in treating cancerous cells. However, colchicine brutally damages a cell’s inner layers by blocking the action of a protein called tubulin. Tubulin plays an important role as cells divide into two. Thus, colchicine effectively halts cell division. Stopping cell division is a vital part of potential cancer drugs. Unfortunately, colchicineattacks not only cancer cells, but healthy ones as well, classifying it as a poison.  One strategy to deal with this has been proposed by Laurence Patterson, Professor of Drug Discovery and Institute Director of the Institute of Cancer Therapeutics at the University of Bradford. Patterson uses a drug delivery system that targets colchicine directly to the target. Many cancers produce a lot of enzymes called matrix metalloproteases (MMPs) which are useful to the cancer because they can disolved extracellular matrix around cells and give the cancer cells access to new blood vessels. Patterson attach long molecular tails to colchicine molecules which target the colchicine directly to the cancer cells. The colchicine is nontoxic until it comes into contact with MMP1, a metalloprotease protein released by tumors. 

Side Effects oc Chemotherapeutic agents

Chemotherapy suffers from problems like drug toxicity and drug resistance (due to increased drug metabolism, increased DNA repair mechanisms and decreased drug import into cells). Although chemotherapeutics and radiation exposure can effectively kill rapidly proliferating tumor cells, the rapidly dividing stem cells of the host’s mucosal epithelium, including the stem cells of the interstinal crypts and epidermis, are also damaged or killed, leading to the clinical condition termed “mucositis”. Oral and gastrointestinal mucositis can be sufficiently painful or debilitating to deter patients from continuing their course of treatment. Cytotoxic chemotherpay is known to cause mucosal injury (MI) both in the oral cavity and to mitotically active intestinal crypt cells. The manifestations of oral mucositis include erythema, ulcer formation, bleeding and exudates. Most of patietns treated for head and neck cancer and almost half of the patients receiving chemotherpay for non-head and neck cancer develop oral complications. The effect of radiation therpay on oral cavity primarily results form local tissue changes. These changes are initiated by a reduction in the prolfieration of basal epithelial cells, causing atrophy. Compromise of the mucosal barrier can also contribue to local invasaion by colonizing microorganisms and, subsequently, to systemic infection (Khan, “Infection and Mucosal Injury in Cancer Treatment, 2001).

Recombinant human keratinocyte growth factor (rhKGF, sold as Kepivance by Amgen, Inc, used as pretreatment, has been shown to cause an increase in measures of mucosal thickness and a 3.5 fold improvement in crypt surfival in the small intestine.

Drugs which Counteract Side-Effects

Granulocyte colony stimulating factor: is used to stimulate neutrophil production in patients udnergoing chemotherapy. Amgen, for example, sells Neupogen® (filgrastim) which is a recombinantly products version of G-CSF and FDA approved for cancer patients receiving myelosuppressive chemotherapy, actue myeloid leukemia patients receiving induction or consolidation chemotherapy, patients udneroing bone marrow transplantation, autologous periopheral blood progenitor cell collection and therapy and patients with severe chronic neutropenia. Sandoz has recentrly received approval for a biosimilar under the provisions of the Biologics Prcie Competition and Innovation Act (BPCIA). 

Treatment of invasive cervical cancer is affected by the stage of the disease, which is based on clincal evaluation.

Chemotherapy: 

Single-agent chemotherpay can be divided into those who received plantinum-based therapy and those who received nonplatinum-based therapy. Cisplatin is regarded as the most active agent in carcinoma of the cervix. Platinum analogs such as Carboplatinhave been investigated in an effort to reduce toxicity.

Combination chemotherapy: 

Combination chemotherapy incldues drugs that have single agent-activity, nonoverlapping toxicity, and additive or synergistic activity with no incrase in toxicity in order to improve response rates and survival. For example, cisplatin has been combined with 5-fluoruracil, bleomycin, ifosfamide, gemcitabine, vinorelbine, paclitaxel, and topotecan in phase II-III trails. These trials showed advantage in response rates when compared with single-agent cisplatin.

Chemoradiotherapy 

Chemoradiotherapy is the treatment of choice for patients with stage IB2 disease (tumour >4cm), invasive carcinoma, confined to cervix). An RCT showed that adding weekly cisplatin to pelvic radiotheraphy before hysterectomy reduced the risk of recurrence of disease and death compared with radiotheraphy and hysterectomy alone.

Three RCTs have shown that improvements in progression free survival and overall survival are greater for chemoradiotheraphy than for radiation alone in patients with locally advanced stage IIB-IVa disease. Platinuum based chemoradiotheraphy is now the standard of care for these patients.

Patients with early stage disease have a high risk of recurrence postoperatively if they have one of the following risk factors: postive nodes, parametrial invasion, or positive surgical margins. Such patients should receive adjuvant cisplatin based chemoradiotheraphy after hysterectomy, as shown by an RCT.

There is a lot of conflicting published work regarding the treatment of builky stage Ib-IIa cervical cancer. While some centers are performing primary surgery as for Ib1 disease followed by tailored postoperative radiation with or without chemotheraphy, the others are in favor of primary chemo-radiation therapy. Neoadjuvant chemotheraphy followed by radical surery has emerged as a possible alternative which may imporve a surfival in patients with stage Ib2 disease.

Concomitant chemoradiation is becoming a new standard in treatment of advanced disease, because it has been clearly shown to improve disease-free, progression-free and overall survival.

The management of recurrent cervix carcinoma has not improved significantly with modern chemotherapy due to several factors. First, carcinoma of the cervix has limited sensitivity to cytotoxic agents, especially when it recurs in the irradiated pelvis. Second, the median response duration is usually 3-70 months. Third, there is a difficulty in showing a meaninful incrase in survival of the patient population as a whole. Fourth, there is refractoriness among patients who have failed any prior chemotherapy.

EGFR Inhibitors:

Activation of EGFR triggers a cascade of events leading to cell reproduction. 

Tarceva (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazo-linamine): has been shown to be useful for treatment of tumors caused by HPV. (see US Patent NO: 6,900,221). 

Radiotheraphy: 

Patients with early stage disease have an intermediate risk of recurrence postoperatively if they have two of the following factors: large tumour size, deep stromal invasion, or involvement of the lymphovascular space. An RCT evaluating 277 women with stage IB disease versus “no further treatment” and at least two risk factors showed that adjuvant radiotheraphy decreased the rate of recurrence and imporved disease free survival.

–Radical hysterectomy:

Radical hysterectomy is the treatment of choice for young healthy patients because it preserves ovarian function. It is thought to be equally effective for patients with early stage disease.

Most often, stage Ib1 cervical cancer is treated by radical hysterectomy with pelvic lymphadenectaomy. Laparoscopically assisted radical vaginal hysterectomy has shown similar efficacy and recurrence rates.

Natural Medicines

Artemisinin: US Patent Publication No. US2007/0142459 discloses methods of treating proliferative cervical disorders by administering a therapeutically effective amount of artemisinin-related compounds. In some embodimetnes, artemisinin or an artemisinin derivative, combined with other anti-viral or anti-cancer therapies such as radiation theraphy are used. Other agents known for their use in the inhibition of cervical cancer include interleukin-2,5′-fluorouracil, nedaplatin, methotrexate, vinblastine, doxorubicin, carboplatin, paclitaxel (Taxol), cisplatin, 13-cis re-moic acid, pyrazoloacridine, and vinorelbine.

Artemisinin (Oinghasosu) is a naturally occuring substance, obtainec by purificatoin from sweet wormwood, Artemisia annua. Artemisinin and its analogs are sesquiterpene lactones with a peroxide bridge. The very low toxicity of these compoudns to humans is a majro benefit.

Formulations of the artemisinin-related compounds suitable for oral administration may be in the form of capsules, pills, pwoders, etc. The topical formulations may include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples include 2-pymolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcholo, dimethyl sulfoxide and azone.

N,N-dimethylglycine: US Patent Publication Number 2006/0183801A1(Assignee, FoodScience, Corporation, Essix Junction, VT) discloses a method of treating, inhibiting the metasis of, or preventing cervical cancer by administering to a patient an effective amount of N,N-dimethylglicine or a pharmaceutically acceptable salt thereof.

See also therapeutic applications of antibodies   See also Antibodies as toxic drug carriers in drug delivery. 

Companies:  Immunogen 

 Often membrane-associated polypeptides are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such polypeptides are more abundantly expressed on the surface of cancer cells. The identification of such tumor associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody based therapies. About 70 humanized monoclonal antibodies (mAbs) against tumor antigens are currently in human clinical trails, and 3 mAbs, Rituximab, herceptin and Alemtuzumab have been approved by the FDA. Such antibodies may also be conjugated to a toxin which can kill the tumor cell. 

Such tumor antigens include receptors of the erbB class such as EGFR, HER2-4, insulin growth factor recetpor I (IGF-RI), c-met, receptors of the tumor necrosis factor family such as DR4-5, DcR1, DCR2, IL-17 recetpor, IL-23 receptor, IL-12 receptor and interferon-alpha receptor (EP2546268).

The mere binding of antibodies to cells does not always lead to their destruction. Thus, attempts have been made to render antibodies cytotoxic by attaching a drug, toxin or radiolabelled isotope to them, so called “magic bullets”. (Segal US 4,676,980) See Drug Delivery

Mechanism of Action

A major antitumor mechanisms of mAbs is thought to be by antibody-dependent cellular cytotoxicity (ADCC). mABs specific for tumor cell surface antigens bind to the tumor cell through their antigen binding domains and, through the Fc region of the mAb, bind to cells such as NK cells and neutrophils that express Fc receptors. This binding event is thought to activate the FcR-expressing cell, which results in ADCC. Most antibodies that mediate ADCC can also cause CDC. However, CDC does not play a big role in the mAb mediated destruction of tumor cells, as these cells tend to overexpress complement regulatoyr proteins (e.g., CD46, CD55, CD59). Interference with the function of these proteins may promote CDC. Therapetic anti-cancer mAbs may also induce apoptosis of cancer cells by blocking binding of growth factors to their receptors and/or sensitive cancer cells to chemotherapy and radiotherapy as well as act as anti-angiogenic agents by blocking the binding of VEGF to its receptor. Therapeutic may also block intracellular signaling pathways. Ionnello (Cancer and Metastusis Reviews 24: 487-499, 2005). For example, bevacizumab binds to the soluble VEGF and thus inhibits angiogenesis, which is important for maintenance of tumor vaculature and tumor health. Goswami (Antibodies 2031, 2, 452-500)

Tumor Antigens and Antibodies which Target These Antigens

B7H4: Terret (WO2009/073533) discloses B7H4 antibodies which are useful for treating cancer. B6-H4 exerts its physiologic function by binding to a receptor on T cells, which in turn induces cell cycle arrest and inhibits the secretion of cytokines, the development of cytotoxicity and cytokine production of CD4+ and CD8+ T cells. Chen (US2008/026235) also discloses administering an effective amount of a B7-H4 antagonist such as an antibody which can be used to enhance an immune response to treat cancer. 

CALLA: The monoclonal antibody J5 is specific for Common Acute Lymphoblastic Leukemia Antigen (CALLA) and can be used to target cells that express CALLA (e.g., acute lymphoblastic leukemia cells).

c-Met: is the cell surface receptor for hepatocyte growth factor (HGF), also known as scatter factor. The c-Met receptor is a disulfide linked heterodimer consisting of extracellular alpha and beta chains. The alpha chain, heterodimerized to the amino terminal portion of the beta chain, forms the major ligand binding stie in the extra cellular domain. HGF binding induces c-Met receptor homodimerization and phosphorylation of two tyrosine residues within the catalytic site, regulating kinase activity. HGF mediated activation of c-Met results in a complex genetic program referred to as invasive growth, consisting of a series of physiological processes, including profliferation, invasion, and angiogenesis that occur under normal physiological conditions during embryonic developmetn and pathologically during oncogenesis. In tumour cells, c-Met activation causes the triggering of a diverse series of signalling cascades resulting in cell growth, proliferation, invasion and protection from apoptosis.

A variety of c-Met pathway antagonists such as monoclonal antibodies with potential clinical applications are currently under clinical investigation. Examples include the anti-c-Met 5D5 antibody generated by Genentech (WO96/38557). WO2009/007427 also describes mosue monoclonal antibodies to c-Met and chimeric variants in which the antigen binding domains of the mosue monoclonal antibody are coupled to the constant region of human IgG1.

Hultberg 9US2012/0156206) discloses a combination of antibodies binding to the human c-Met protein and more specifically combinations of two or more c-Met antibodies which bind to distinct, non-overlapping epitopes. Such antibodies have advantageous properties in that the combinations can produce potent inhibition of HGF independent activaiton of the human c-Met receptor.

CD4: An anti-CD4 Mab that is useful for T cell depletion therapy is IDEC-151 (US 6,136,310). 

CD20: In 1997, the FDA approved RITUXAN® (IDEC Pharmaceuticals Corp. and Genetechn, Inc.,) (also referred to as rituximab), a chimeric anti-CD20 antibody for the treatment of non-Hodgkin’s lymphoma.

Rituximab is a mAb consisting of a human kappa constant region, a human IgG Fc portion and a murine variable region. The exact antitumor mechanisms of rituximab remains unclear, but it is assumed that it exerts its effects by various mechanisms including activaiton of complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC) and signaling of apoptosis by binding to the CD20 antigen specifically expressed on B cells. Although most patients with B cell non-Hodgkin lymphoma respond to initial rituximab treatment, a significant number become unresponsive. It has been shown that complement regulatory proteins such as CD55 and CD59 are associated with acquisition of resistance. (K. Takei, Leukemia Research 30 (2006) 625-631. 

CD22: is a 140 kDa member of the Siglec family of cell surface proteins that is expressed by most mature B cell lineages. . It is also a member of the sialadhesin family of adhesion molecules that includes sialoadhesin and myelin-associated glycoprotein. Sialoadhesin and CD22 mediate cellular interactions by recognizing sepcific cell surface sialylated glycoconjugates. Remarkably, mAbs which blocked CD22 ligand binding were shown to accelerate mature B cell turnover and inhibit the survival of adoptively transferred normal and malignant B cells in vivo. In addition, mAbs that boudn CD22 ligan binding domains induced significant CD22 internalization, depleted marginal zone B cells and reducd mature recirculating B cell numbers by 75-85% in the mouse (Haas, J Immunol. 2006, 177(5): 3063-73). 

CD22 is an attractive molecular target because of its restricted expression. It is not exposed on embvyonic stem cells but is highly expressed on B cells in NHL. A murine anti-CD22 mAb has been developed for imaging and treatment of NHL. Anti-CD22 antiboides also have potential as carriers of cytotoxic agents since they rapidly internalise and human CD22 has been trageted using mAb conjugates linked to toxins such as derivatives of Pseudomonas exotoxin. A particularly exciting possibility is using CD22 targetted RNAses. This has the advantage of overcoming limitations experienced in using toxins. RNAses are not immunogenic in animal studies nor does a naked RNAse cause immunological problems in humans (newton, Expert Opin. biol. Ther: (2001) 1(6): 995-1003).

Jones (US 8,389,688) discloses humanized versions of anti-CD22 mouse mAbs which can be used in the treatment of B cell malignancies.

Epratuzumab is a humanized antihuman CD22 IgG1 antibody which has shown promising clinical activity both as a single agent and in combination with rituximab in patients with non-Hodkin’s lymphomas (NHL) Wang, Clin Cancer Res. 2003, 9 (10 Pt2): 3982S-90S

CD33: The monoclona antibody MY9 is a murine IgG1 antibody that binds specifically to the CD33 antigen and be used to target cells that express CD33 (e.g., acute myelogenous leukemia (AML) cells. 

CD88: IDEC-114 is an anti-CD80 MAb for treating autoimmune diseases and preventing organ transplant rejection (US 6,113,898). 

CD200: Bowdish (WO2007/084321) discloses anti CD200 anitobides for treatment of cancer. 

EGFR: Aberrant expression of epidermal growth factor receptor (EGFR) has been observed in a variety of solid tumors, and often correlates with a poor clinical outcome for patients. These features make EGFR an important target for cancer therapeutis, linke mAbs and tyrosine kinase inhibitors (TKI). Anti-EGFR mAbs paneitumumab (Vectibix) and cetuximab (Erbitux) are established agents in the treatment of colorectal cancer. Yang, “Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency” mAbs (2015). 

IGF-IR (type 1 insuline-like growth factor receptor): has been implicated in promoting oncogenic transformation, growth and survival of cancer cells. WO20007/110339 discloses antibodies that bind to the extracellular domains of IGF-IR and inhibit receptor activation. 

VEGF (Human vascular endothelial growth factor): VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors as well as intracoular disorders. VEGF is a homodimeric glycoprotein that has been isolated from several sources. It shows highly specific mitogenic activity for endothelial cells. It ahs important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life. Anti-VEGF nuetralizing antibodies suppress the growth of a variety of human tumor cell lines in mice. WO 94/10202, WO 98/45332, WO 2005/00900 and WO 00/35956 refers to antibodies against VEGF.

Humanized monoclonal antibody bevacizumab (sold under the trade name Avastin) is an anti-VEGF antibody used in tumor therpay (WO 98/45331).

Ranibizumab (trade name Lucentis) is a monoclonal antibody fragment derived form the same parent murine antiboy as bevacizumab. It is much smaller than the parent and has been affinity matured to provide stronger binding to VEGF-A (WO 98/45331). It is an anti-angiogenic that has been approved to treat the “wet” type of age-related macular degneration, a common form of age related vision loss. 

Another anti-VEGF antiboy HuMab G6-31 is described in US 2007/0141065. 

ANG-1/ANG-2 (Human angiopoietin-2): ANG-1 and ANG-2 were discovered as ligands for the Iites, a family of tyrosine kinases that is selectively expressed within the vascular endothelium. Corneal angioenesis assay have shown that both ANG-1 and ANG-2 had similar effects, acting syntergistically with VEGF to promote growth of new blood vessels. 

WO2011/117329 discloses a bispecific, bivalent antibody that includes a first antigen bidning site for human VEGF and a second antigen binding ste for human ANG-2. 

Histones: It has been shown that extracellular histones released in response to inflammatory challenge are mediators contributing to endothelial dysfunction, organ failure and death during spesis. Esmon (US2009/0117099) discloses that inhibitors of histones such as antiboides that bind to H1, H2A, H2B, H3 and H4 can also be used to alleviate toxicity in cancer cuased by chemotherapy, radiation and cytokines.  Svanborg (US2006/0233807) also disclsoes using a moeity which specifically binds to hisone such as an antibody such that the chromatin assembly or remodeling activity of the cell is inhibited. The method is disclosed as being useful for the treatment of cancer. 

Antigens which are Overesxpressed in Certain Types of Cancers and Monoclonal Antibodies which Target these antigens

Claudin-18.2: is overexpressed in up to 80% of gastrointestinal adenocarcinomas and 60% of pancreatic tumors, in addition to other solide cancers. Astellas has a monoclonal candicate IMAB362 that targets this tight junction protein.

Antibodies against Immune check point proteins

Immuen checkpoint proteins are a set of proteins that act as either stimulators or inhibitors of the immune system. By dampening or attenuating the “off-switch” an immune response is enhanced. The most well known checkpoint targets are the following:

cytotoxic T lymphocyte-assocaited antigen 4 (CTLA-4):  ipilimumab is an anti-CTLA-4 soled as Yervoy by Bristol-Myers Squibb. (WO2016063263) discloses that the bacterium Bacteroides fragilis improved the antitumor efficacy of an anti-CTLA-4 antibody. 

programmed cell death 1 (PD-1):  Pembrolizumab is an anti-PD-1 sold as Keytruda by Merck

programmed death ligand 1 (PD-L1). nivolumab is sold as Opdivo by Bristol-Myers Squibb.  (US20150352206) discloses that Bifodobacterium improved the antitumor efficacy of an anti-PD-L1 antibody in a mouse model of melanoma. 

T cell receptor therapeutics:

TCR arm – antibody domain; TCR like antibodies:

As part of normal immune surveillance, intracellular.proteins are degraded by the proteasome into short peptide fragments of about 8-10 amino acids which are presented on the cell surface bound to MHC molecules, known as human leukocyte antigen (HLA) molecules in humans. Circulating T cells bind specific peptide MHC complexes with their TCRs, continuously monitoring for intracellular ref flags that are processed and displayed on the cell surface. Immunocor’s tebentafusp is approved by the FDA for uveal melanoma. The bispecific biologic molecules combines a TCR arm that recognizes tumour cells with an antibody domain to bind and activate T cells. For tebentafusp, Immunocore uses a TCR domain to recognize an epitope of gp100, a transmembrane glycoprotein that is located on the ER and epxressed in melanocytes and melanoma –as presented on HLA-A*02:01. The T cell engaing arom fo the bispecifiv binds CD3on T cells. For this therapy, one just needs ti know gp100 (a tumor specific antigen) and the patien’ts HLA. (Nature Reviews Drug Discovery, 21, May 2022, 321). 

Roche has made it into the clinic with a TCR mimicking bespecific called RG6007. One antibody domain recognizes a fragment of the cancer associated transcription factor WTI presented on HLA-A&*02 and the other binds CD3 on T cells. (Nature Reviews Drug Discovery, 21, May 2022, 321)

HLA restriction poses one challenge for TCR based approaches. There are multiple HLA genes, and each has numerous variants. But first generation TCR therapies can only bind theri targets in patients harbouring a certain HLA allele. Some alleles are more common in particular populations, providing starting points for frug developers. Tebentafusp, for example, takes aim at the HLA-A*02 subtype, which is present in about 40% of the global population and over 95% of white populations. But patients with other subtypes will need different therapeutics. A greater challenge for teh field is choosing the best TCR target. Finding a protein expressed exclusively in tumour cells is just the begninng, as MHC presentation of the peptidome is a complex and dynamic process. (Nature Reviews Drug Discovery, 21, May 2022, 321)

T cell redirecting antibody (TRAB):

Antibodies which bind a tumor cell specific antigen on one arm and CD3 on a T cell by the other arm thereby inducing cytotoxicity against the tumor cell by bridging the tumor cell and the T cell have been developed. (“Proprietary Innovative antibody engineering technologies in Chugai Pharmaceutical”, 12/18/2012). 

MedLinePlus

Frederick National Laboratory

Unpredictability in Cancer Treatment

Non-correlation between In Vitro and In Vivo treatment: Cancer treatment is highly unpredictable. For example, even though the EGFR was identified in some cancers as a drug target, the in vitro effectiveness of a drug in inhibiting the EGFR turned out to be a poor proxy for how effective that drug actually was in treating cancer in vivo. Numerous EGFT inhibitors that showed promising in vitro activity failed for a variety of reasons. These included poor pharmacokinetics due to poor adsorption or rapid metabolism (or both), undersirable drug-drug ineractions, drug toxicity due to drug binding onto healthy cells, sdrug toxicity due to binding onto other receptors and metabolite toxicity. OSI Pharmaceuticals v. Apotex (USPTO, No: IPR2106-01284)

Challenges with Experimental Models: Despite some successes, many cancer still have a high mortality rate and no effective treatment. For many cancers, survival rates have not changed in decades – pancreatic cancer remains almost 100% lethal, and the oeverall survival rate for lung cancer has improved only from 13 to 16 %. The key difficulty is that cancer is a complex and heterogenous disease: many genes are amplified, deleted, mutated, and up- or down-regualted. Many pathways are activated or suppressed. These chances vary substantially in different cancers, in different patients with the same cancer, and even in different tumor samples from the same patient (Fortney, 2011, p. 465, last ¶). Although mice are crucial for cancer research, cancer is a complex, three-dimensional disease that changes, evolves and spreads through the body. Mice, easy to breed and genetically manipulate, and with a completed genome sequence, are the obvious choice for scientists who wanted to pick apart these processes and test new drugs. Bust despite having broad similarities, mice have significant differences. For one, most mouse trmours originate in different types of tissues from humans and unlike humans, their lethalaly cells can maintain the ends of their chromosomes, a key factor influencing which mutations tumour cells develop. One of the key mouse models, involves grafting human cancer cells into mice and seeing how the resulting tumours respond to treatment. But human cells are likely to behave differently in a mouse than in a human body, making results hard to interpret (Carina Dennis, Nature, (2006), pp. 739-741)

Combination Therapy and Dosing

It is recognized in the art that a benefit of using chemotherpaeutic agents in conjunction with other therpaies, e.g., an antibody antagonist, can be useful for allowing adminsitraiton of lower dooses of chemotherpaeutic agetns, thereby potentially resulting in a reduction in toxic side effects. It is also known in the art that therpaeutically effective dosages of chemotherpaeutic agents can vary when these drugs are used in treatment combiantions. Methods for experimetnally determining therapeutically effective dosages of chemotherapeutic drugs and other agents for use in combination treatment regimends are descried in the literature. For example, the use of metronomic cheotherapy dosing, i.e., providing more frequent lower doses in order to minimize toxic side effects, has been described extensively in the literature. A combiantion treatment regimen encompases treatment regimens in which administration of a chemothreapetuic agent is initiated prior to, during, or after treatment with the second agent, e.g., an antibody, and continues until any time duringtreatment with the other agent or after termination of treatment with the other agent. It It also includes treatment in which the agents being used in combiantnion are adminsitered simultaenoeusly or at differnt times and/or at decreasing or increasing intervals during the treatment period. (Van Epps (US 2007/0048325).

Cancer Treatment Strategies

Surgical debulking: If surgical removal is possible, this is a favored treatment strategy. However, surgery may miss micrometastases.

Chemotherapy: Most, if not all, chemotherapeutic agents kill cancer cells through the induction fo apoptosis. Cisplatin, for example, is extensively used for the treatment of a broad spectrum of turmors. Chemotherapy suffers from problems like drug toxicity and drug resistance (due to increased drug metabolism, increased DNA repair mechanisms and decreased drug import into cells). It also has devastating side effects such as gut-tering vomitus and diarrhea, alopecia, and fatal vulnerability to infections. Cisplatin, for example, has preogressive, irreversible side effects including nephrotoxicity and ototoxicity. Evidence indicates that cisplatin ototoxicity is closely related to the increased production of reactive oxygen species (ROS).

Tumor Immunology: Tumor cell destruction should be possible by the immune system. Strategies along this line of thought include the following:

(i) Cancer Vaccines refers to therapeutic immunization in patients with cancer, with the main aim of breaking tolerance toward self-tumor-associated antigens. One strategy is to pulse antigen presenting cells such as dendritic cells (CDs) with tumor antigens in the hope that that such DCs will induce host protective and therapeutic antitumor immunity. These approaches are currently limited for clinical application because few human tumor antigens on a tumor cell that can activate the immune system have been found. In the best studied human melanoma where a class of tumor associated proteins have been identified, it is unclear which of the identified tumor associated Ags is the best choice to induce effective tumor rejection in vivo or how effective they are.

The advent of massively parallel sequences has now made it possible to sequence the entire gehome of exome (coding regions) of tumor and matched normal cells to identify all of the muations that have occurred. The subset of those mutations that alters protein coding sequences creates personal, novel antigen “neoantigen” which may provide the “foreign” signal needed for cancer immunotherpaty This is important because the use of self antigens which are selectively expressed or overexpressed in tumors requires overcoing both central tolerance (whereby autoreactive T cells are deleted in the thymus during development) and periopheral tolerance (whereby mature T cells are suppressed by regulatory mechnisms). (Hacohen, “Getting Persoanl with Neoantigen-based therpaeutic cancer vaccines” DOI: 10 (2013).

Another interesting therapeutic approach is the use of peptides as vaccines. Phage-display libraries are panned against therapwutic antibodies. Peptides identified that resemble the original antigen to which the antibody binds have been called “mimotopes”. The resulting mimotpe may be used to elicit humoral and cellular respones. (Brissette “Identificaiton of cancer targets and therapeutics using phage display” Current Opinion in Drug Discovery and Development 2006, 9(3) 363-369).

(ii) Introduce genes encoding MHC Ags, toll-like receptor agonists, costimulatory molecules or cytokines into tumor cells. The goal here again is to improve the immunogenicity and the Ag-presenting capability of tumor cells. For example, DCs can be raised from bone-marrow stem cells upon culture with cytokine combinations. During the differentiation culture they can be transfected with recombinant viral vectors encoding cytokines like IL-12, IL-7, or IL-2. If such cells are then injected intratumorally they capture antigen, migrate to draining lymph nodes and orchestrate a potent CTL response. Artificial expression of the transgene greatly enhances the antitumor immune response.

Interestingly, most of the pulsing has been done with peptides rather then proteins (which would require DCs to process the antigen). This is because when take DCs and put them into plastic culture dishes, giving them cytokines, etc., this matures them. So most of the DCs become mature. One way to counteract this is to do this in vivo. For example, FLT3L is a way to increase DCs in vivo to increase DCs prior to taking out the blood to grow the DCs in culture. Morese et al, JCO, 2001 found that if they systemically gaive Flt3 ligand to mice, DC population increased. Then use IL-14, GM-CSF and TNFa for growing the DCs (CD34+ HPC) in vitro.

Patients given subcutaneous GM-CSF administration increase mobilization of CD80+ DCs. Studies have shown that just giving GM-CSF is therapeutic. Newer treatments include taking tumor cells from patients, growing them, transfecting them with plasmid encoding with GMCF, then irradiating them to use them as a vacine. Trials have shown this stimulated DCs leading to increased CD8+ cells at tumor sites.

Another approach is to use TLR agonists to activate local immune response. For example, the TLR7 agonist, imidazoquinolines and TLR 9 CpG are being used to activate the innate immune system.

(iii) Direct Tumor Transfection with Co-stimulatory Molecules: Another strategy is to take the tumor and turn it into an APC. Take tumor and put in rv-B7.1 to induce T cell activation and induce tumor recognition. Researchers have found that by doing this they were able to generate gp100- and MARt-1-specific T cells found in most A2+ patients.

(iv) Adoptive immunotherapy: (CD4+ or CD8+ cells): Adoptive immunotherpay of cancer refers to a therapeutic approach in which immune cells with an anti-tumor reactivity are adminstered to a tumor bearing host, with the aim that the cells mediate, either directly or indirectly, the regression of an established tumor. Transfusion of lymphocytes, particularly T lymphocytes, falls into this category and investigators at the National Cancer Institute (NCI) have used autologous reinfusion of peripheral blood lymphocytes or tumor infiltrating lymphocytes (TIL) T cell cultures from biopsies of subcutaenous lymph nodules, to treat several human cancers (US 4,690,914). For example, T cells that have a natural or genetically engineered reactivity to a patients’ cancer are expanded in vitro using a variety of means and then adoptively transferred into the cancer patient.One approach with respect to T helper cells is to adoptively transfer T helper cells to initiate CTL immunity. Infusion of CD4+ T cell clones specific for P815AB has shown tumor destruction through a CD8 mediated destruction. Other reports have shown that such transferring induces a broad immune response. For example, TIL expanded in vitro in the presence of IL-2 have been adoptively transferred to cancer patients, resulting in tumor regression in select patients with metastatic meloma. Melanoma TIL grown in IL2 have been identified as activated T lymphocytes CD3+ HLA DR+, which are predominantly CD8+ cells with unique in vitro antitumor properties. Many long term melanoma TIL cultures lyse autologous tumors in a specific MHC class 1 and T cell antigen receptor dependent manner.

It has been deomonstrated that CD8+ cytotoxic T lymphocytes (CRLs) recognize epitope peptides derived from tumor-associated antigens (TAAs) presented on MHC class I molecules and lyse tumor cells. Since the discovery of the MAGE family as the first example of TAAs, many other TAAs have been discovered using immunological apporaches. Some of the discovered TAAs are now in clinical development as targets of immunotherapy. TAAs discovered so far include MAGE, gp100, SART, and NY-ESO-1. For a listing of human tumor antigens recognized by T cells see (Cancer Immunol Immunother (2001) 50:3-15).

Chimeric antigen receptor T-cell (CAR-T) therapies:

CAR T cells combine the specificity of a mAb with the cytolytic capacity of a CAR T cell. This is achieved by fusing the scFv of a mAb (or another antigen recognition domain) to a transmembrane domain and intravellular signaling domains capable of elicity a T cell response. CARs that contain only the CD3-epsilon endodomain are known as first generation CARS. those that contan one costimulatry domain (such as CD28 or 41BB) are known as second-generation CARS; and those that contain two or more costimulatory domains are known as third generation CARS. To date, most clinical experience and sucess has been amassed with CAR T cells targeting CD19, a surface protein involved in B cell signlaing that is expressed on B cell malignancies. Given that its expression is restricted to the B cell lineage, and patients can live without healthy B cells, CD19 has emerged as a promising target for CAR T cell immunotherapy. Other targets that have demonstrated clinical sucess in B cell malignancies include CD22 fo B-ALL and BCMA for multiple myeloma. T date, most CAR T cell trials have used autologous T cells for transduction. A cancer patient’s T cells are collected, activated with antibodies or antibody coated beads, and then transduced, most commonly with a lentivirus or retrovirus, to express the CAR molecule. CAR T cells are then expanded in vitro to sufficient numbers to infuse back into the pateint. Patiens often recieve lympho depleting chemotherapy before T cell infusion. Majzner (“clinical lessons learned from teh first leg of the CAR T cell journey, Nature Medicine, 25, pp. 1341-1355, 2019)

Numerous factors can affect the potency and quality of a CART-T therpay including the production process (e.g., CD4/CD8 T cell ratios, T cell phenotype, levels of non-transduced cells, duration of activation, transgene construct (high or low epression, insolators, etc), vector choic (retroviral, adenoviral or transposon), CAR design (e.g., scFv affinity, stability and immunogenicity, spacer lenght and signaling domains) and input donar blood cells (e.g., starting cell number and exposure to different treatments and conditioning regimens). (Nature Biotechnology, 35(10), October 2017.

–Kymriah: which is developed by Novartis was approved by the FDA for treating relapsed B cell acute lymphoblastic leukemia in 25 and under.

–Yescarta, made by Kite Pharma, a Gilead Science company for example has been approved for treatment of certain types of non-Hodgkin lymphomoa in which patient’s white blood cells are extracted, modified, and then injected back into the patient. It was shown to cure 36% of patients completely and to reduce tumors in 82% of patients. It costs about $373,000.

Cancer Specific Antibodies: See outline

T Cell Activation with Treg Depletion: Knutson et al. 2005. Treg cells are known to express CTLA-4. Prudhomme G, J Luekocyte Biol, 2004 have given mice anti-CTLA4 and vacine against gp100. However, side effect get large autoimmunity.

Induce apoptosis.

   –TNF, FasL and TRAIL:

In models utilizing animals engrafted with human tumors, treatment with TRAIL/Apo2L induces significant tumor-specific apoptosis, tumor regression, and improved survival with no identifiable toxicity. TNF, LT-alpha1Beta2, FasL, and TRAIL are all expressed and used to kill cancer cells by professioanl cytotoxic cells, such as CD8+ CTLs and NK cells. In addition, Fasl, TNF, and TRAIL are expressed by activated CD4+ T cells, B cells and macrophages. Human immature DCs also express these TNF family ligands on their cell membrane. Cell surface expression and secretion of these cytotoxic ligands appears to be tightly regulated and differentiation stage-dependent with the dendritic cell lineage. The possible regualtory mechanism might include activiteis of cytokines and/or metalloproteases.

Complement factor or receptor antagonists: Goldenberg (US2006/0140936) discloses multispecific antagonists that react specifically with at least one complement factor or complement regulatory protein in the thearapy of various inflammatory diseases including cancer. In preferred embodiments, the complement factor includes C3, C5, C3a, C3b and C5a.

Medof (US12/920293) disclose that administration of a C3aR antagonist and a C5aR antagonist such as an an antibody to C3aR and an antibody to C5aR induced apoptosis in a cancer cell expressing a c3a recetpor (C3aR) and a C5a receptor (C5aR). Lambris (US2011/0044983A1) also discloses treating cancer by admistering complement inhibitors such as inhibiotrs of C3aR and C3aR.

Corticosteroids: are used in the treatment of patients with blood disorders such as multiple myeloma. corticosteroids may work by causing programmed cell death of certain cells. Corticosteroids are also used in the treatment of cancer because they decrease inflammation. This cn decrease swelling around tumors in the spine and brain.

Induce phagocytosis. Cells undergoing apoptosis express specific signals, including lipids like phosphatidylserine (PS) that facilitate recognition and ingestion by macrophages. The possibility exists that one might be able to stimulate phagocytosis of tumor cells in the absence of a death signal to thereby delete cancer cells without even having any associated bystander effects observed during conventional chemotherapeutic treatment which is based on the induction of apoptosis.

Attract DCs to tumors with Chemokine: For example, injection of the chemokine CCL20 attracts DCs into the tumor, leading to induction of anti-tumor response. Intratumoral CpG can also activate tumor DC, leading to induciton of systemic anti-tumor immunity.

Counteracting Angioenesis: Counteracting angioenesis hampers tumor growth and spread. Angiogenesis involved the action of endogenous growth hormones such as vascular endotherlial growth factor (VEGF-A/B, VEGF-C and/or VEGF-D). Thus, counteracting the action of such growth factor indirectly counteracts tumor growth because angiogenesis is at least in part prevented. Placental growth factor (PIGF) is also involved in angiogenesis. Whereas PIGF is primarily involved in angiogenesis in tumour tissue, VEGF plays an important role in angiogenesis in toehr (non-tumour) tissue as well.

Tumour laser thermotherapy: has been shown to be superior to surgical exicsion, partly due to the laser induced immunological effect such as increased number of tumour infiltrating macrophages and CD8 lymphocytes (Ivarsson, British J. of Cancer, 2005, 93, 435-440).

–Photodynamic therapy (PDT): is a systemic administration of tumor localizing photosensitzers and subsequent irradiation with light of the appropriate wavelenght. The combination of drug uptake in maliganant tissues and selective delivery of laser generated light provides effective therapy, with efficient tumor cytotoxicity and minimal normal tissue damage. Activated photosensitivers interact with molecular oxygen to produce singlet oxygen that destroys neoplastic cells with minimal normal tissue dage. PDT utilizing the hematoporphyrin derviative (Hpd) has been used clincially for palliation of obstructive lesions of the esophagus, and the tracheobronchial tree, for treatment of bladder tumors and for loca control of various tumors on the skin surface (Canti, Anti-Cancer Drugs 1994, 5, 443-447). Photodynamic therapy is a chemotherapeutic approach that utilizes a bifunctional reagent that localizes to the target tissue relative to the surrounding tissue and is toxic to the arget tissue when epxosed to light.

Inhibition of Detrimental Cytokines: It is now accepted that inflammation is a driving force behind cancer and reflects that the inflammation is a protective attempt to remove the injury. However, disease progressio in cancer is dependent on the coplex interaction beween the tumor and the host inflammatory response. There is substantial evidence in advanced cancer that host factors such as weight loss, poor performance status and the host systemic inflammatory response are linked. For example, elevated level of C-reactive protein is now included in the definition of “cachexia” as a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass. The inflammatory cytokines that have been involed in wasting diseases are IL-6, TNF-alpha, IL-1beta and interferon-gamma (Argiles, European J Pharmacology 668, 2011, S81-S86. IL-6 psotive staining in carcinoma of the esophagus and also been assocaited with shorter survival (Chen, Mol Cancer, 2013, 12:26).

Immunotoxins: In this treatment an anti-tumor cell antibody is used to delvier a toxin to the tumor cells. However, in common with chemotherpaeutic approaches, immunotoxin therapy also suffers from drawbacks when applied to solid tumors. Tumor mass is generally impermeable in macromolecular agents such as antibodeis and immunotoxins. (Thorpe (US 2006/0228299)

Vasculature targetting: A promosing treatment strategy is to target the vasculature of solid tumors. Targeting the blood vessels of the tumors, rather than the tumor cells themselves, has certain advantages in that it is not likely to lead to the development of resistant tumor cells, and the targeted cells are readily accessible. Thorpe (US 2006/0228299)

Thorpe (US 2006/0228299) discloses that phosphatidylerine (PS) is a specific marker of tumor vasculature which has led to the development of new anti-PS immunoconjugates for delivering anti-cellular agents, toxins and coagulation factors to tumor blood vessels. Other examplary phosphatidylservine binding prtoeins includes annexins such as annexin V.

Precision Cancer Treatment:

Researchers can now sequence a person’s entire genome in mere days. Doing such sequencing can often point treatment i the right direction becasue they can reveal genetic mutations in genes that may be important in the type of cancer. Knowing the specific type of cancer can lead to personalized treatment for this type of cancer.

Treatment of Cancer Side Effects:

Pegfilgrastim-apgf injection is used to treat neutropenia (low white blood cells) that is caused by cancer medicines. It is a synthetic (man-made) form of a substance that is naturally produced in your body called a colony stimulating factor. Pegfilgrastim-apgf helps the bone marrow to make new white blood cells. This medication is usually given at least 24 hours after chemotherapy to stimulate the growth of new, healthy, white blood cells (WBC). Pegfilgrastim is a longer acting form of filgrastim and the manufacturer recommends that it should not be given within 14 days prior to chemotherapy.