초록 ▼

PD-L1과 CD3를 동시에 타겟하여 PD-L1/PD-1 면역관문을 저해함과 동시에 T 세포와 암세포의 인공적인 면역시냅스를 유도하는 ACE-05 이중항체에 대한 최종개발후보 확보를 위해 ACE-05 및 대조 항체의 시료 생산 및 분석, ACE-05 작용기전 및 in vitro 효능 연구, 면역인간화 마우스에서의 효능 평가, hCD3e TG 마우스를 이용한 효능 평가와 cytokine/TIL 분석, 그리고 설치류와 원숭이에서의 PK와 예비독성 평가를 완료/확인하였음
1. 항체 시료 생산 및 분석: 재조합 항체(Anti-PD-L

PD-L1과 CD3를 동시에 타겟하여 PD-L1/PD-1 면역관문을 저해함과 동시에 T 세포와 암세포의 인공적인 면역시냅스를 유도하는 ACE-05 이중항체에 대한 최종개발후보 확보를 위해 ACE-05 및 대조 항체의 시료 생산 및 분석, ACE-05 작용기전 및 in vitro 효능 연구, 면역인간화 마우스에서의 효능 평가, hCD3e TG 마우스를 이용한 효능 평가와 cytokine/TIL 분석, 그리고 설치류와 원숭이에서의 PK와 예비독성 평가를 완료/확인하였음
1. 항체 시료 생산 및 분석: 재조합 항체(Anti-PD-L1 mAb, anti-CD3 mAb, BiTE-05, ACE-05) 4종을 동물세포 임시발현을 통해 확보하고, 정제된 단백질에 대한 순도 및 LC-MS를 이용한 hetero-tetrameric 구조 확인
2. 작용기전 및 in vitro 효능연구: ACE-05의 작용기전에 대해 분자이미징 기법을 통해 관찰하고, PBMC, CD3+ T cell, 그리고 CD8+ T cell을 활용하여 이중항체의 효능을 확인
3. 면역인간화 마우스에서의 효능 평가: PBMC humanized 마우스 폐암(NSCLC) 모델에서 ACE-05의 유효 농도 파악과 NSCLC 이외 다른 PD-L1 양성 암에서의 ACE-05의 효능확인
4. hCD3e TG 마우스 모델에서의 효능 평가 및 cytokine/TIL 분석: S.C, orthotopic 마우스 모델을 이용하여 ACE-05의 항암효과 확인 및 cytokine/TIL 분석 완료
5. 설치류와 영장류에서의 PK 분석: Rat, cynomolgus monkey 모델에서 ACE-05의 BiTE-05 대비 설치류 약 3배, 영장류 약 3.5배 높은 혈중안정성 확인
6. 영장류에서 cytokine 분석: Cynomolgus monkey에서 ACE-05 단회 투여 후 Th1/Th2 cytokine level 분석을 통한 적은 side-effect 가능성 확인

(출처 : 요약서 3p)

Abstract ▼

Patients with stage IIIB or higher, who account for more than 60% of lung cancer patients, have a very poor prognosis with a survival rate of less than 6%. Platinum-based doublet ortriplet chemotherapy, a traditional standard treatment for non-small cell lung cancer (NSCLC), which accounts for 85% o

Patients with stage IIIB or higher, who account for more than 60% of lung cancer patients, have a very poor prognosis with a survival rate of less than 6%. Platinum-based doublet ortriplet chemotherapy, a traditional standard treatment for non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancer, has severe side effects and resistance. Targeted Drugs of tyrosine kinase inhibitor (TKI), a representative of targeted therapy, have a response rate of 20 to 25% in adenocarcinoma patients, but have no efficacy in other NSCLC patients. Anti-PD-1 or anti-PD-L1 antibody therapeutics, recently FDA-approved immune checkpoint inhibitors, show superior efficacy and treatment persistence to other drugs.
However, they have just 30% overall response rate (ORR) in NSCLC patients. Other immuno-oncology drugs such as T cell bispecific technology, represented by BiTE, and CAR-T technology have higher ORR and complete response (CR) over 60%, but they are highly toxic and very expensive, and yet have not been developed in NSCLC. In order to improve theunmet needs of BiTE, our company has developed a novel format of T cell bispecific antibody and named it as Antibody Like Cell Engager (ALiCE). ALiCE preserves F(ab’)₂ structure of the parental antibody to maintain high binding affinity to the surface target ofcancer cell and substitutes the Fc region with a monovalent Fv to induce immune response.
This 2-by-1 format of bispecific antibody is expected to be more productive and more serum-stable than BiTE due to its antibody-like shape and higher molecular weight, respectively.

In this study, our aim is to develop the non-clinical candidate of ACE-05, an ALiCE-based PD-L1×CD3 bispecific antibody. To this end, we analyzed basic characteristics of ACE-05, investigated the mechanism of action, evaluated the anti-tumor efficacy in vitro and in vivo models with cytokine analysis, and accessed PK and toxicity in rat and cynomolgus monkeymodels.

First, ACE-05 and control antibodies necessary for PoC research were produced in HEK293F cells, and then purified through KappaSelect or anti-CH1 bead to obtain a sufficient amount of sample. The purified ACE-05 protein was confirmed by SDS-PAGE, capillary electrophoresis (CE), SEC-HPLC, and CEX-HPLC analyses to confirm the purity and structural binding characteristics. In addition, thermal stability and stability to pH and room temperature storage were evaluated, and disulfide bond formation of ACE-05 were confirmed through LC-MS analysis.

In order to understand the mechanism of action of ACE-05, Biacore 8K confirmed that the binding affinity of ACE-05 to PD-L1 was more than 300 times higher than that of CD3. As a result, ACE-05 showed higher PD-L1-dependent T cell activation and cytotoxicity against cancer cells than BiTE, whereas in the absence of PD-L1, T cell activation and immune cytokine levels of ACE-05 were very low compared to BiTE. It indicates that high cytotoxicity activity and low toxicity of ACE-05 may be due to unique structure of ALiCE.

In PBMC-reconstituted NSG mouse model bearing HCC827 NSCLC tumor, tumor growth inhibition of ACE-05 was very effective up to 0.05 mg/kg as similar to BiTE. However, in the body weight change as an indicator of toxicity, BiTE had a large change at 0.5 mpk, and many mice died, whereas the body weight change after treatment of ACE-05 was relatively low, and almost no mice were killed, implicating low toxicity of ALiCE.

In order to evaluate the tumor growth inhibition of ACE-05 in various cancer model, PBMC-reconstituted NSG mice carrying MDA-MB231 tumor and HCT116 colorectal tumor were used. However, unexpectedly, the reconstruction of PBMC in such mice did not occur and the number of T cells was very few. It may caused by low cell-to-cell engager effect of the T cell bispecific antibodies. As a result, both ACE-05 and BiTE had poor tumor growth inhibition.

On the other hand, in the hCD3e TG mouse model with an intact immune system, ACE-05 confirmed remarkable tumor growth inhibition and low toxicity. In the case of BiTE, the mice were all dead at early time implying high toxicity. The cytokine analyses also confirmed the high PD-L1-specific and low non-specific activity of ACE-05 compared to BiTE.

PK analysis performed in rodents and primates showed ACE-05 had longer serum half life by approximately 3 times and 3.5 times in rodents and primates, respectively, compared to BiTE-05.

In this study, we confirmed that unique structure of ACE-05 led PD-L1-dependent strong cancer cell killing but, at the same time, reduced toxicity. Therefore ALiCE technology may be a tool to resolve the unmet medical needs of anti-PD-1 or anti-PD-L1 antibodies and BiTE T cell engagers.

(출처 : SUMMARY 6p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 6
• CONTENTS ... 8
• 목차 ... 9
• 제1장 연구개발과제의 개요 ... 10
• 제1절 연구 개발의 목적 ... 10
• 제2절 연구 개발의 필요성 ... 11
• 제3절 연구 개발의 범위 ... 14
• 제2장 국내외 기술개발 현황 ... 17
• 제1절 국내 동향 ... 17
• 제2절 국외 동향 ... 18
• 제3장 연구개발수행 내용 및 결과 ... 20
• 제1절 주요성과 ... 20
• 제2절 연구개발 수행 내용 ... 22
• 제4장 목표달성도 및 관련분야에의 기여도 ... 78
• 제1절 목표 달성도 ... 78
• 제2절 관련 분야 기여도 ... 79
• 제5장 연구개발결과의 활용계획 ... 80
• 제1절 연구개발결과의 활용계획 및 추가연구의 필요성 ... 80
• 제2절 사업화 전략 및 방법 ... 81
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 82
• 제7장 연구시설·장비현황 ... 83
• 제8장 참고문헌 ... 84
• 끝페이지 ... 86