“The sad fact is that around 70% of cancer patients with solid based tumors don’t respond to classical chemotherapy,” said Joseph Tabernero, MD, PhD, head of Val d’Hebron Institute of Oncology in Spain at the recent Avacta London Science Day.
According to Tabernero, this is why immunotherapeutics which encourage the immune system to recognize the tumor and destroy tumor cells have become more widely used. He states: “Check point inhibitors impact the cancer immunity cycle, offering a better solution and a longer term response.”
Amrik Basran, PhD, CSO of Avacta agreed, saying, “Standard chemotherapy for solid tumors has a poor survival rate. Immunotherapy using PD1 or PD-L1 check point inhibitors offer better survival compared to standard chemotherapy as these allow T cells to recognize and destroy tumor cells. Combination immunotherapies of checkpoint inhibitors mAbs offer even better survival.
“However, combination immunotherapy is expensive as it combines two mAbs, which individually are costly to produce and can give better efficacy for the patient. We need to look at producing safer medicines that are more effective for the patient but are also cost-effective to produce such as bispecifics and drug conjugates. Bispecifics are potentially good therapeutics as they bind to two different antigens involved in the disease and are more effective than a monotherapy, but should be cheaper to produce.”
Small but potent
Basran explains that Avacta’s technology platform, known as an Affimer®, is based on naturally occurring human protein (StefinA), which is engineered to display two loops forming a binding surface and can be used as the building blocks to generate bispecifics and drug conjugates. According to Basran, Affimers are antibody mimemtics consisting of a single polypeptide chain of around 14 kDa in size, which do not contain any disulphide bonds or glycosylation. Affimers are around a tenth of the size of most therapeutic mAbs and even when formated to extend their half-lives should have better tissue and tumor penetration than antibodies. They are generated using large phage display libraries that are screened for high affinity binders. Basran says, “Their small size and simple structure means they can be produced by a range of expression systems. Also, because they are small and very soluble this potentially makes them easier to formulate at high concentrations.”
To substantiate these assertions, Basran presented data on Avacta’s AVA04-251, a PD-L1 binding Affimer which is attached via G4S linkers to a human IGg1 Fc region. This data showed that the molecule is expressed from Expi HEK and CHO cells during transient expression at 250 mg/L and has a half-life of ~120 hours in mouse PK models. He also demonstrated that the Affimer-Fc could be stored at 200 mg/mL in a simple buffer such as phoshate buffered saline (PBS) at +4°C with ~5% aggregate formed after 7 months as seen in samples analyzed by SEC-HPLC.
Basran comments: “We see a small amount of aggregation with AVA04-251 but this is without any formulation work and is very encouraging for long-term storage at high concentrations. We are planning to do viscosity testing at these high protein concentrations to determine if the Affimer-Fc could in the future, be dosed sub-cutaneously in patients.”
To indicate the in vitro and in vivo effectiveness of AVA04-251, Basran presented preclinical data which showed that AVA04-251 Fc generates T-cell activation comparable to PD-L1 blocking mAb, Avelumab in Staphylococcus enterotoxin B (SEB) and mixed lymphocyte reaction (MLR) assays. He also showed that in a mouse hPD-L1 MC38 model, AVA04-251 Fc (dosed at 10 mg/kg) slowed down tumor growth, which was compareable to the anti-tumor effect seen with PD-L1 binding mAb, Atezolizumab dosed at 5 mg/kg in this mouse model.
Affimers can also be used as a platform to generate a range of different types of bispecific antibody and antibody mimetics which Basran says “have the potential for better synergistic anti-tumor efficacy.” Basran detailed Avacta’s proof-of-concept molecule Avastin-AVA04-251, which combines the anti-VEGF antibody, Avastin genetically fused to AVA04-251 to the end of the heavy chain. He also discussed AVA21, a bispecific which combines two AVA04 PD-L1 binding Affimers linked to a human IGg Fc region and two other immune checkpoint receptor Affimers (AVA19) for lymphocyte activation gene 3 (LAG-3). “Blocking of LAG-3 on T cells helps to maintain the immune system’s attack and relieves T-cell exhaustion,” Basran states.
Avacta’s Affimers are versatile and can also be used as drug conjugates and are currently being assessed for their anti-tumour activity using the tumor microenvironment activate drug conjugate platform (TMAC) in collaboration with scientists at Tufts University. These Affimer TMACs circulate as one molecule, in which the drug conjugate is inactive. When the Affimer binds to tumor cells bearing PD-L1, for example, the Affimer TMAC molecule is delivered into the the tumor microenvironment. Here the drug conjugate comes into contact with enzymes which are expressed in tumors, and the drug is released, allowing tumors to become receptive to immunotherapy, which in this case is delivered by the anti-PD-L1 Affimer. This Affimer targeted approach should also make using these toxic drugs safer and more effective for the patient.
William Bachovchin, PhD, professor at Tufts University School of Medicine says, “We have an engineered TMAC which includes Avacta’s PD-L1 Affimer attached to a Fibroblast Activation Protein (FAPα) linker and this is attached to an I-DASH Inhibitor drug, which we’re working with.” He then presented details to show that the FAPα enzyme is selectively overexpressed in the tumor microenvironment of many solid tumors including breast, pancreatic, liver, lung, and ovarian tumors. Bachovin says, “FAPα enyme activity is over 100-fold above background in many different tumors so that a linker recognized by this protein is a good target for enzymatic cleavage to release toxic compounds inside tumors.”
He explains that I-DASH is a good choice for a drug conjugate adding, “I-DASH induces pyroptosis so it punches holes in macrophage cells acting like nature’s trip wire to release proinflammatory cytokines and other antitumor immune signals which alert the T-cell troops that these cancer cells need to be destroyed.”
Bachovin presented syngeneic mouse data that showed AVA-04 182-Fc (an Fc formatted bivalent Affimer that binds to mouse PD-L1) when dosed at 10 mg/kg and combined with the I-DASH inhibitor in a MB49 mouse urothelial carcinoma model, reduced tumor volume growth over 25 days, with multiple animals having fully regressed tumors by end of the study. He also showed similar tumor reduction and animal survival results from a second study with AVA-04 251-Fc, (an Fc formatted, bivalent Affimer that binds to human PD-L1) when dosed at 10 mg/kg and combined with the I-DASH inhibitor) in a “hPD-L1” MC38 mouse colon adenocarcinoma model with human PD-L1 knock-in over 13 days. In both studies, the Affimer/I-DASH combination therapy showed similar anti-tumor activity to Avelumab (dosed at 10 mg/kg) and Atezolizumab (dosed at 5 mg/kg) mAb and I-DASH combinations.
Bachovin comments: “These results are promising because it means I-DASH in combination with a mAb or an Affimer improves anti-tumor activity. The I-DASH enhances the checkpoint inhibitors effectiveness. In our studies, this meant several animals dosed with Affimer-I-DASH combination didn’t die and by marshalling the immune system were effectively cured, ensuring the tumor didn’t come back.”
Alastair Smith, PhD, Avacta’s CEO summarized: “The Affimer platform has lots of potential and can be used in a number of ways in place of traditional mAb therapeutics. The major advantages Affimers have when compared to mAbs is that they are smaller so can penetrate tumors more effectively and we have good proof of concept data for this. Additionally, as they are small they could also be manufactured in microbial systems such as E.coli or Pichia more cost effectively and because they are not mAbs there are also fewer IP costs involved.”
Smith concludes: “Our key challenge is to focus on one area as we have limited resources and so we are developing our TMACs first as we have some promising preclinical data with Tufts. Also, our partnership deals to develop Affimer drug candidates with biotechs like LG Chem Life Sciences and Moderna are providing good external validation and endorsement for our technology, which is giving us the right trajectory for adoption and deals with larger, more risk averse biopharmas.”