WO2022089601A1

WO2022089601A1 - Bifunctional fusion protein consisting of il-2 and antibody subunit

Info

Publication number: WO2022089601A1
Application number: PCT/CN2021/127523
Authority: WO
Inventors: 傅阳心; 彭华; 曹帅帅
Original assignee: 中国科学院生物物理研究所
Priority date: 2020-10-30
Filing date: 2021-10-29
Publication date: 2022-05-05
Also published as: CN114437228B; CN114437228A

Abstract

Provided in the present invention are a bifunctional fusion protein consisting of IL-2 and an antibody subunit, and the use thereof in the preparation of an anti-tumor drug. The bifunctional fusion protein comprises a heterodimer or a homodimer.

Description

一种IL-2与抗体亚单位构成的双功能融合蛋白A bifunctional fusion protein composed of IL-2 and antibody subunits

技术领域technical field

本发明属于生物医药技术领域，具体的，涉及一种IL-2与抗体亚单位构成的双功能融合蛋白。The invention belongs to the technical field of biomedicine, and specifically relates to a bifunctional fusion protein composed of IL-2 and antibody subunits.

背景技术Background technique

白细胞介素2(IL2)又称为T细胞生长因子，发现于1976年，在体外实验中刺激T细胞克隆扩增[1]。IL2为四个α螺旋的糖蛋白，分子量为15.5Kd[2]，主要由活化的CD4 T细胞分泌，其它细胞如活化的CD8细胞、NK细胞、NKT细胞以及ILCs细胞也能产生少量的IL2[3,4]。IL2以自分泌和旁分泌途径结合于受体细胞上发挥功能，对T、NK细胞具有重要的调节作用。Interleukin 2 (IL2), also known as T cell growth factor, was discovered in 1976 to stimulate T cell clonal expansion in vitro [1]. IL2 is a four α-helix glycoprotein with a molecular weight of 15.5Kd[2]. It is mainly secreted by activated CD4 T cells. Other cells such as activated CD8 cells, NK cells, NKT cells and ILCs cells can also produce a small amount of IL2[2]. 3,4]. IL2 binds to receptor cells through autocrine and paracrine pathways to function, and plays an important role in regulating T and NK cells.

IL2的受体由IL2Rα(CD25)，IL2Rβ(CD122)和IL2Rγc(CD132)三个亚基构成，IL2与三种亚基之间的亲和力存在差异[5,6]。IL2与α亚基之间低亲和力结合(Kd～10-8M)，与βγ二聚体中等亲和力结合(Kd～10-9M)，与αβγ三聚体高亲和力结合(Kd～10-11M)[7,8]。The receptor of IL2 consists of three subunits: IL2Rα (CD25), IL2Rβ (CD122) and IL2Rγc (CD132). There are differences in the affinity between IL2 and the three subunits [5,6]. IL2 binds with low affinity to α subunit (Kd～10-8M), binds with moderate affinity to βγ dimer (Kd～10-9M), and binds to αβγ trimer with high affinity (Kd～10-11M)[7 ,8].

不同亲和力受体在不同细胞上的表达水平存在差异：βγ受体主要表达在静息状态的T细胞、CD8 T记忆细胞和NK细胞上；α受体在细胞活化后表达上调，活化后的T细胞表达高亲和力的αβγ受体。Treg细胞持续高表达CD25分子，Treg细胞对IL2分子具有很高的亲和力[9]。由于受体表达的差异性，低剂量的IL2优先扩增表达高亲和力受体的Treg细胞，高剂量的IL2才能有效活化CD8 ⁺T细胞和NK细胞[10]。 There are differences in the expression levels of different affinity receptors on different cells: βγ receptors are mainly expressed on resting T cells, CD8 T memory cells and NK cells; α receptors are up-regulated after cell activation, and activated T cells Cells express high-affinity αβγ receptors. Treg cells continue to highly express CD25 molecules, and Treg cells have high affinity for IL2 molecules [9]. Due to differences in receptor expression, low doses of IL2 preferentially expand Treg cells expressing high-affinity receptors, and high doses of IL2 can effectively activate CD8 ⁺ T cells and NK cells [10].

IL2与三聚体受体的亲和力最高，而三聚体受体主要在Treg细胞上持续表达而仅仅在活化的效应细胞上短暂上调表达；临床上使用低剂量的IL2会优先扩增Treg细胞，而使用高剂量的IL2才能活化效应T细胞。高剂量的IL2会导致更大的毒副作用而限制了IL2的使用。IL2 has the highest affinity for the trimeric receptor, and the trimeric receptor is mainly continuously expressed on Treg cells and only temporarily up-regulated on activated effector cells; clinical use of low doses of IL2 will preferentially expand Treg cells, The use of high doses of IL2 to activate effector T cells. High doses of IL2 can lead to more toxic side effects and limit the use of IL2.

为了克服这一问题，尝试通过不同的手段对IL2分子进行改造，使其降低对Treg的结合而提高对效应T细胞和NK细胞的结合来增强其在肿瘤治疗中的效果，同时避免高剂量使用而导致的毒性。目前对IL2的改造有以下几种方法：In order to overcome this problem, we tried to modify the IL2 molecule by different means to reduce the binding to Treg and increase the binding to effector T cells and NK cells to enhance its effect in tumor therapy, while avoiding the use of high doses resulting toxicity. At present, there are several methods for the transformation of IL2:

(1)PEG化的IL2(1) PEGylated IL2

IL2的分子量为15.5KD，在体内血清中的半衰期为10-85min，为了达到治疗效果需要多次重复给药。PEG化的IL2提高了IL2的总体分子量延长了IL2的半衰期，同时通过PEG化修饰位点的不同可以使IL2偏向于结合IL2-Rα或者IL2-Rβ[11]。The molecular weight of IL2 is 15.5KD, and the half-life in serum in vivo is 10-85min. In order to achieve the therapeutic effect, repeated administration is required. PEGylated IL2 increases the overall molecular weight of IL2 and prolongs the half-life of IL2. At the same time, IL2 can be biased to bind to IL2-Rα or IL2-Rβ through different PEGylation sites [11].

(2)IL2/aIL2抗体复合物(2) IL2/aIL2 antibody complex

IL2与aIL2抗体复合物的结合一方面可以延长IL2的半衰期，另一方面由于aIL2抗体可以与IL2分子不同部位的结合，使IL2/aIL2抗体复合物表现出与受体CD25或者CD122结合的不同倾向性。On the one hand, the combination of IL2 and aIL2 antibody complexes can prolong the half-life of IL2. On the other hand, because aIL2 antibodies can bind to different parts of the IL2 molecule, the IL2/aIL2 antibody complexes show different tendencies to bind to receptors CD25 or CD122. sex.

(3)IL2突变改造(3) IL2 mutation transformation

通过对IL2上面不同受体结合位点的点突变改造，也可以改变IL2与不同受体亚基的亲和力。通过对IL2分子L80F、R81D、L85V、I86V和I92F五个碱基的点突变，使IL2发生构象改变，增加与CD122的结合同时使下游信号的转导不依赖于CD25分子。突变的IL2偏向于扩增CD8+T细胞和NK细胞，具有更低的毒副作用并且在小鼠的肿瘤模型中表现出更好的肿瘤治疗效果[12]。The affinity of IL2 to different receptor subunits can also be changed by point mutation engineering of different receptor binding sites on IL2. Through the point mutation of five bases of IL2 molecule L80F, R81D, L85V, I86V and I92F, the conformation of IL2 is changed, the binding to CD122 is increased, and the downstream signal transduction is independent of CD25 molecule. Mutated IL2 is biased towards the expansion of CD8+ T cells and NK cells, has lower toxic side effects and shows better tumor therapeutic effects in mouse tumor models [12].

在我们实验室之前的改造中，我们对IL2同时进行了降低CD25亚基结合(F42A)和增加CD122亚基结合的突变。这种双突变的IL2分子比单独的CD25亚基亲和力下降或者单独的CD122亚基亲和力上升的突变都具有更好的治疗效果[13]。双突变的IL2不仅在MC38肿瘤模型中具有很好的治疗效果；而在TUBO等冷肿瘤模型中，与TKI或者7.16.4抗体的联合治疗也具有很好的治疗效果。说明在增加CD122亚基亲和力的同时进一步降低CD25亚基的亲和力可以更进一步提高IL2的治疗效果。In a previous modification in our laboratory, we mutated IL2 to both decrease CD25 subunit binding (F42A) and increase CD122 subunit binding. This double-mutated IL2 molecule has a better therapeutic effect than either the CD25 subunit alone or the CD122 subunit alone with increased affinity [13]. Double-mutated IL2 not only has a good therapeutic effect in the MC38 tumor model, but also has a good therapeutic effect in combination with TKI or 7.16.4 antibody in cold tumor models such as TUBO. It shows that while increasing the affinity of CD122 subunit, further reducing the affinity of CD25 subunit can further improve the therapeutic effect of IL2.

上述针对IL2的种种改造都是通过降低IL2对CD25分子的结合降低或者对CD122亚基的结合增加。虽然这一改造的策略相比于野生型的IL2分子的治疗效果有提升，但是仍不能完全消除瘤内Treg细胞对治疗效果的抑制作用。如何进一步比较和分析瘤内Treg细胞和效应T细胞上CD25以及CD122受体表达的差异性来进一步提高IL2治疗效果仍是一个关键的问题。 All of the above-mentioned modifications to IL2 are achieved by reducing the binding of IL2 to the CD25 molecule or increasing the binding of the CD122 subunit. Although this modified strategy improved the therapeutic effect of wild-type IL2 molecules, it still could not completely eliminate the inhibitory effect of intratumoral Treg cells on the therapeutic effect. How to further compare and analyze the differences in the expression of CD25 and CD122 receptors on intratumoral Treg cells and effector T cells to further improve the therapeutic effect of IL2 is still a key issue .

TNF家族的共刺激或共抑制性分子也在Treg细胞上高表达，是调节Treg细胞功能的靶点分子，常见的分子有ICOS、GITR、OX40、4-1BB等。The co-stimulatory or co-inhibitory molecules of the TNF family are also highly expressed on Treg cells and are target molecules that regulate the function of Treg cells. Common molecules include ICOS, GITR, OX40, and 4-1BB.

一些共刺激分子如GITR和OX40分子在瘤内Treg上高表达，在活化的CD4和CD8 T细胞上也上调表达。OX40分子表达于瘤内的T细胞上，而且在瘤内的Treg上高表达；全身给予激活性aOX40抗体后可以导致瘤内Treg数目和比例的下降而发挥抗肿瘤的作用[14]。在aOX40抗体的临床试验中未观察到明显的Treg数目下降，但是能观察到效应T细胞的数目上升[15]。Some co-stimulatory molecules such as GITR and OX40 are highly expressed on intratumoral Treg and also up-regulated on activated CD4 and CD8 T cells. The OX40 molecule is expressed on T cells in the tumor, and it is highly expressed on the Treg in the tumor; systemic administration of activating aOX40 antibody can lead to a decrease in the number and proportion of Treg in the tumor and play an anti-tumor effect [14]. In clinical trials of aOX40 antibodies, no significant decrease in the number of Treg was observed, but an increase in the number of effector T cells was observed [15].

OX40分子共同表达于效应T细胞和Treg细胞上，目前使用的OX40抗体发挥抗肿瘤作用可以既发挥活化效应细胞又发挥删除Treg细胞作用。OX40发挥抗肿瘤作用依赖于瘤内CD4 T和CD8 T细胞的扩增，而且治疗后小鼠具有免疫记忆re-challenge后肿瘤不会再复发[16]。另一方面，小鼠实验中使用的OX40抗体OX86也可以发挥删除Treg的功能，这种删除作用依赖于活化性的FcγR而不依赖于抑制性的FcγRIIB。而且将OX86的rIgG1Fc分别换成小鼠的IgG2a和IgG2An297A(不与FcR结合)后，发现IgG2a的Fc具有更好的肿瘤控制能力，说明在此肿瘤模型上OX40抗体功能的发挥主要依赖于删除Treg的功能；而且删除作用主要发生在Treg细胞，而非Treg的CD4 T细胞以及CD8 T细胞的数目和绝对数并未下降[17]。在使用OX40靶点进行治疗时，我们可以利用Treg和效应T细胞上OX40表达丰度的不同，对不同T细胞群发挥不同作用。除了OX40抗体单独使用外，OX40分子还可以同化疗、放疗以及其它细胞因子联合使用，都具有协同作用而发挥更好的抗肿瘤作用[18]。The OX40 molecule is co-expressed on effector T cells and Treg cells. The currently used OX40 antibody can play an anti-tumor effect, which can both activate effector cells and delete Treg cells. The anti-tumor effect of OX40 depends on the expansion of intratumoral CD4 T and CD8 T cells, and the tumor will not recur after the mice have immune memory re-challenge after treatment [16]. On the other hand, the OX40 antibody OX86 used in mouse experiments can also delete Tregs, and this deletion depends on activating FcγR but not on inhibitory FcγRIIB. Moreover, after replacing the rIgG1Fc of OX86 with mouse IgG2a and IgG2An297A (not binding to FcR), it was found that the Fc of IgG2a has better tumor control ability, indicating that the function of OX40 antibody in this tumor model mainly depends on the deletion of Treg Moreover, the deletion mainly occurred in Treg cells, and the number and absolute number of non-Treg CD4 T cells and CD8 T cells did not decrease [17]. When using OX40 targets for treatment, we can take advantage of differences in the abundance of OX40 expression on Treg and effector T cells to exert different effects on different T cell populations. In addition to the single use of OX40 antibody, OX40 molecules can also be used in combination with chemotherapy, radiotherapy and other cytokines, all of which have synergistic effects and play a better anti-tumor effect [18].

因此，IL2/aOX40双功能分子，以及CTLA4和其他TNF家族的共刺激或共抑制性分子ICOS、GITR、4-1BB等的抗体与IL-2细胞因子构成的融合蛋白双功能抗体，在临床上是非常具有开发潜力的肿瘤治疗新方案。Therefore, IL2/aOX40 bifunctional molecules, as well as CTLA4 and other TNF family co-stimulatory or co-inhibitory molecules ICOS, GITR, 4-1BB, etc. antibodies and IL-2 cytokines constitute fusion protein bifunctional antibodies. It is a new tumor treatment solution with great potential for development.

发明内容SUMMARY OF THE INVENTION

本发明首先涉及一种双功能融合蛋白，所述的融合蛋白为异源二聚体，其特征在于，The present invention first relates to a bifunctional fusion protein, the fusion protein is a heterodimer, and is characterized in that:

所述的异源二聚体包括：Described heterodimer includes:

(1)白介素2(IL-2)与免疫球蛋白Fc单链连接而成的异源二聚体第一单体；(1) The first monomer of a heterodimer formed by linking interleukin 2 (IL-2) and immunoglobulin Fc single chain;

(2)抗CTLA4分子或TNF家族的共刺激或共抑制性分子的抗体的Fab或ScFv与免疫球蛋白Fc单链连接而成的异源二聚体第二单体；(2) A heterodimeric second monomer formed by linking the Fab or ScFv of an antibody against a CTLA4 molecule or a co-stimulatory or co-inhibitory molecule of the TNF family and an immunoglobulin Fc single chain;

所述的第一单体与第二单体通过Fc单链的二聚化连接，构成所述的异源二聚体；The first monomer and the second monomer are connected by dimerization of the Fc single chain to form the heterodimer;

所述的CTLA4或TNF家族的共刺激或共抑制性分子包括但不限于：OX40、4-1BB、ICOS、GITR。The co-stimulatory or co-inhibitory molecules of CTLA4 or TNF family include but are not limited to: OX40, 4-1BB, ICOS, GITR.

所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；The immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain with ADCC effect knocked out through gene mutation;

优选的，所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；更优选的，所述的免疫球蛋白Fc单链为人IgG的Fc单链。Preferably, the immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain with ADCC effect knocked out through gene mutation; more preferably, the immunoglobulin Fc single chain is human Fc single chain of IgG.

所述的CTLA4或TNF家族的共刺激或共抑制性分子为OX40、4-1BB，所述的抗CTLA4的抗体(aCTLA4)或抗TNF家族的共刺激或共抑制性分子的抗体为抗OX40的抗体(aOX40)、抗4-1BB的抗体(a4-1BB)；The co-stimulatory or co-inhibitory molecules of the CTLA4 or TNF family are OX40, 4-1BB, and the antibody of the anti-CTLA4 antibody (aCTLA4) or the co-stimulatory or co-inhibitory molecule of the anti-TNF family is anti-OX40 Antibody (aOX40), anti-4-1BB antibody (a4-1BB);

所述的第二单体中，In the second monomer,

所述的抗体的Fab为人源化抗体的Fab或全人化抗体的Fab；The Fab of the antibody is the Fab of a humanized antibody or the Fab of a fully humanized antibody;

所述的抗体的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；The ScFv of the antibody is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;

更优选的，所述的第二单体为：More preferably, the second monomer is:

抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体的单体，所述抗体的单体包含一条轻链和一条重链；优选的，所述抗体为人源化抗体或全人抗体。Monomer of an antibody against CTLA4 or an anti-TNF family of costimulatory or co-inhibitory molecules, the antibody monomer comprises one light chain and one heavy chain; preferably, the antibody is a humanized antibody or a fully human antibody.

更优选的，所述的异源二聚体包括：More preferably, the heterodimer includes:

(1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus:

1)序列如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) The wild-type IL-2 protein whose sequence is shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, the mutant comprising any one or any combination of the following mutation sites ;F42A, L80F, R81D, L85V, I86V and I92F;

2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) necessary connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;

3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) The Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the knob mutation shown in SEQ ID NO.4 type Fc, or hole mutant Fc as shown in SEQ ID NO.5;

(2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises:

1)序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL和序列如SEQ ID NO.8所示的抗OX40抗体重链VH&CH1组成的抗OX40抗体Fab区；1) the anti-OX40 antibody Fab region composed of the anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7 and the anti-OX40 antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.8;

或：2)序列如SEQ ID NO.9所示的抗OX40单链抗体(ScFv)：Or: 2) anti-OX40 single chain antibody (ScFv) whose sequence is shown in SEQ ID NO.9:

和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 Knob mutant Fc, or hole mutant Fc as shown in SEQ ID NO.5;

第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;

第二单体：其为：Second monomer: which is:

(1)序列如SEQ ID NO.11所示的抗OX40抗体VH-CH1-Fc(knob)和序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL组成的第二单体；(1) a second monomer composed of an anti-OX40 antibody VH-CH1-Fc (knob) whose sequence is shown in SEQ ID NO.11 and an anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7;

或(2)序列如SEQ ID NO.12所示的多肽(aOX40 ScFv-Fc(knob))。Or (2) a polypeptide whose sequence is shown in SEQ ID NO. 12 (aOX40 ScFv-Fc(knob)).

本发明还涉及一种双功能融合蛋白，所述的融合蛋白为同源二聚体，其特征在于，The present invention also relates to a bifunctional fusion protein, the fusion protein is a homodimer, and is characterized in that:

所述的同源二聚体的单体为：The monomer of the homodimer is:

一分子白介素2(IL-2)与一分子抗OX40抗体Fab通过任意方式连接构成的单体，或，A molecule of interleukin 2 (IL-2) and a molecule of anti-OX40 antibody Fab are linked in any way to form a monomer, or,

一分子白介素2(IL-2)与一分子抗OX40单链抗体(ScFv)通过任意方式连接构成的单体。A monomer composed of a molecule of interleukin 2 (IL-2) and a molecule of anti-OX40 single-chain antibody (ScFv) linked in any way.

优选的，所述的同源二聚体的单体自N端开始依次包含：Preferably, the monomers of the homodimer include sequentially from the N-terminus:

(1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；(1) The wild-type IL-2 protein shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites ;F42A, L80F, R81D, L85V, I86V and I92F;

(2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；(2) necessary connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;

(3)抗OX40抗体的Fab或ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；(3) Fab or ScFv of anti-OX40 antibody; the Fab is the Fab of a humanized antibody or the Fab of a fully humanized antibody, and the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;

(4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。(4) Fc of the antibody; the Fc of the antibody is a fully human wild-type Fc or a No-ADCC mutant Fc.

更优选的，所述的同源二聚体的单体为：More preferably, the monomer of described homodimer is:

(1)SEQ ID NO.13(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、(1) SEQ ID NO. 13 (aOX40-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

(2)SEQ ID NO.14(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))所示序列。(2) The sequence shown in SEQ ID NO. 14 (IL2-aOX40: IL2-VL-VH(ScFv)-Fc(wt)).

本发明还涉及另一种双功能融合蛋白，所述的双功能融合蛋白为抗CTLA4抗体与IL2的组合成的双功能融合蛋白，所述的融合蛋白为异源二聚体；The present invention also relates to another bifunctional fusion protein, the bifunctional fusion protein is a bifunctional fusion protein composed of an anti-CTLA4 antibody and IL2, and the fusion protein is a heterodimer;

所述的异源二聚体包括：Described heterodimer includes:

1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) a wild-type IL-2 protein as shown in SEQ ID NO.1, or a mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites; F42A, L80F, R81D, L85V, I86V and I92F;

3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) Fc single chain of IgG as shown in SEQ ID NO.2, or Fc of No-ADCC mutant IgG as shown in SEQ ID NO.3, or knob mutant as shown in SEQ ID NO.4 Fc, or a hole mutant Fc as shown in SEQ ID NO.5;

1)序列如SEQ ID NO.21所示的抗CTLA4抗体轻链VL-KCL与序列如SEQ ID NO.22所示的抗CTLA4抗体重链VH&CH1组成的抗体Fab区；1) the antibody Fab region composed of the anti-CTLA4 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.21 and the anti-CTLA4 antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.22;

或：2)序列如SEQ ID NO.23所示的抗CTLA4单链抗体(ScFv)：Or: 2) anti-CTLA4 single-chain antibody (ScFv) whose sequence is shown in SEQ ID NO.23:

第二单体：其为：Second monomer: which is:

(1)序列如SEQ ID NO.24所示的多肽(aCTLA4 VH-CH1-Fc(knob))和序列如SEQ ID NO.21所示的抗CTLA4抗体轻链组成的第二单体；或(1) a second monomer composed of a polypeptide (aCTLA4 VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.24 and an anti-CTLA4 antibody light chain whose sequence is shown in SEQ ID NO.21; or

(2)序列如SEQ ID NO.25所示的多肽(a CTLA4 ScFv-Fc(knob))。(2) The polypeptide (a CTLA4 ScFv-Fc(knob)) whose sequence is shown in SEQ ID NO.25.

所述的同源二聚体的单体为：The monomer of the homodimer is:

一分子白介素2(IL-2)与一分子抗CTLA4抗体Fab通过任意方式连接构成的单体，或，A molecule of interleukin 2 (IL-2) and a molecule of anti-CTLA4 antibody Fab are linked in any way to form a monomer, or,

一分子白介素2(IL-2)与一分子抗CTLA4单链抗体(ScFv)通过任意方式连接构成的单体。A monomer composed of a molecule of interleukin 2 (IL-2) and a molecule of anti-CTLA4 single-chain antibody (ScFv) linked in any way.

(3)抗CTLA4抗体的Fab或ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；(3) Fab or ScFv of anti-CTLA4 antibody; the Fab is the Fab of a humanized antibody or the Fab of a fully humanized antibody, and the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;

(1)SEQ ID NO.26(aCTLA4-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、(1) SEQ ID NO.26 (aCTLA4-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

(2)SEQ ID NO.27(IL2-a CTLA4：IL2-VL-VH(ScFv)-Fc(wt))所示序列。(2) The sequence shown in SEQ ID NO.27 (IL2-a CTLA4: IL2-VL-VH(ScFv)-Fc(wt)).

本发明还涉及另一种双功能融合蛋白，所述的双功能融合蛋白为抗4-1BB抗体与IL2的组合成的双功能融合蛋白，所述的融合蛋白为异源或同源二聚体；The present invention also relates to another bifunctional fusion protein, the bifunctional fusion protein is a bifunctional fusion protein composed of anti-4-1BB antibody and IL2, and the fusion protein is a heterologous or homodimer ;

所述的异源二聚体包括：Described heterodimer includes:

1)序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链与序列如SEQ ID NO.30、31所示的抗4-1BB抗体重链VH&CH1组成的抗体Fab区；1) the antibody Fab region composed of the anti-4-1BB antibody light chain whose sequence is shown in SEQ ID NO.28 and 29 and the anti-4-1BB antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.30 and 31;

或：2)序列如SEQ ID NO.32、33所示的抗4-1BB单链抗体(ScFv)：Or: 2) anti-4-1BB single chain antibody (ScFv) whose sequence is shown in SEQ ID NO.32, 33:

第二单体：其为：Second monomer: which is:

(1)序列如SEQ ID NO.34、35所示的多肽(a4-1BB VH-CH1-Fc(knob))和序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链组成的第二单体；或(1) The composition of the polypeptide (a4-1BB VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.34 and 35 and the light chain of anti-4-1BB antibody whose sequence is shown in SEQ ID NO.28 and 29 the second monomer; or

(2)序列如SEQ ID NO.36、37所示的多肽(a4-1BB ScFv-Fc(knob))。(2) The polypeptide (a4-1BB ScFv-Fc(knob)) whose sequence is shown in SEQ ID NO.36 and 37.

所述的融合蛋白为同源二聚体，其特征在于，Described fusion protein is homodimer, it is characterized in that,

所述的同源二聚体的单体为：The monomer of the homodimer is:

一分子白介素2(IL-2)与一分子抗4-1BB抗体Fab通过任意方式连接构成的单体，或，A molecule of interleukin 2 (IL-2) and a molecule of anti-4-1BB antibody Fab are linked in any way to form a monomer, or,

一分子白介素2(IL-2)与一分子抗4-1BB单链抗体(ScFv)通过任意方式连接构成的单体。A monomer composed of a molecule of interleukin 2 (IL-2) and a molecule of anti-4-1BB single-chain antibody (ScFv) linked in any way.

所述的同源二聚体的单体自N端开始依次包含：The monomers of the homodimer, starting from the N-terminus, sequentially include:

(3)抗4-1BB抗体的Fab或ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；(3) Fab or ScFv of anti-4-1BB antibody; the Fab is the Fab of a humanized antibody or the Fab of a fully humanized antibody, and the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;

更优选的，所述的同源二聚体的单体为：如More preferably, the monomer of described homodimer is: as

SEQ ID NO.38(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 38 (a4-1BB(LOB12.3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.39(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 39 (a4-1BB(3H3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.40(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 40 (IL2-a4-1BB(LOB12.3): IL2-VL-VH(ScFv)-Fc(wt)),

SEQ ID NO.41(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示序列。The sequence shown in SEQ ID NO. 41 (IL2-a4-1BB(3H3):IL2-VL-VH(ScFv)-Fc(wt)).

本领域技术人员同样也应当明确：Those skilled in the art should also be clear:

使用如：Use as:

SEQ ID NO.52-55所示的氨基酸序列编码的抗人OX40抗体的功能结构The functional structure of the anti-human OX40 antibody encoded by the amino acid sequence shown in SEQ ID NO.52-55

SEQ ID NO.56-57所示的氨基酸序列编码的抗人CTLA4抗体的功能结构The functional structure of the anti-human CTLA4 antibody encoded by the amino acid sequence shown in SEQ ID NO.56-57

SEQ ID NO.58-61所示的氨基酸序列编码的抗人4-1BB抗体的功能结构The functional structure of the anti-human 4-1BB antibody encoded by the amino acid sequence shown in SEQ ID NO.58-61

替换上述各个双功能融合蛋白的相似功能嵌段，同样可以达到本发明所述的技术效果。Replacing the similar functional blocks of each of the above-mentioned bifunctional fusion proteins can also achieve the technical effect of the present invention.

本发明还涉及编码所述异源二聚体和同源二聚体的核苷酸序列。The present invention also relates to nucleotide sequences encoding said heterodimers and homodimers.

优选的，preferably,

编码所述异源二聚体第一单体的核苷酸序列为SEQ ID NO.15(IL2-Fc(hole))所示的核苷酸序列；The nucleotide sequence encoding the first monomer of the heterodimer is the nucleotide sequence shown in SEQ ID NO.15 (IL2-Fc(hole));

编码所述异源二聚体第二单体的核苷酸序列为：The nucleotide sequence encoding the second monomer of the heterodimer is:

SEQ ID NO.16(aOX40：VL-KCL)、SEQ ID NO. 16 (aOX40:VL-KCL),

SEQ ID NO.17(aOX40：VH-CH1-Fc(knob))、SEQ ID NO. 17 (aOX40: VH-CH1-Fc(knob)),

SEQ ID NO.18(aOX40 ScFv-Fc(knob))、SEQ ID NO. 18 (aOX40 ScFv-Fc(knob)),

SEQ ID NO.42(a4-1BB(LOB12.3)：VL-KCL)、SEQ ID NO.42 (a4-1BB(LOB12.3):VL-KCL),

SEQ ID NO.43(a4-1BB(3H3)：VL-KCL)、SEQ ID NO.43 (a4-1BB(3H3):VL-KCL),

SEQ ID NO.44(a4-1BB(LOB12.3)：VH-CH1-Fc(knob))、SEQ ID NO. 44 (a4-1BB(LOB12.3): VH-CH1-Fc(knob)),

SEQ ID NO.45(a4-1BB(3H3)：VH-CH1-Fc(knob))、SEQ ID NO. 45 (a4-1BB(3H3): VH-CH1-Fc(knob)),

SEQ ID NO.46(a4-1BB(LOB12.3)：ScFv-Fc(knob))、SEQ ID NO. 46 (a4-1BB(LOB12.3): ScFv-Fc(knob)),

SEQ ID NO.47(a4-1BB(3H3)：ScFv-Fc(knob))所示的核苷酸序列；The nucleotide sequence shown in SEQ ID NO.47 (a4-1BB(3H3): ScFv-Fc(knob));

编码所述同源二聚体的核苷酸序列为：The nucleotide sequence encoding the homodimer is:

SEQ ID NO.19(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 19 (aOX40-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.20(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 20 (IL2-aOX40: IL2-VL-VH(ScFv)-Fc(wt)),

SEQ ID NO.48(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 48 (a4-1BB(LOB12.3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.49(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 49 (a4-1BB(3H3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.50(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 50 (IL2-a4-1BB(LOB12.3):IL2-VL-VH(ScFv)-Fc(wt)),

SEQ ID NO.51(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示的核苷酸序列。The nucleotide sequence shown in SEQ ID NO. 51 (IL2-a4-1BB(3H3):IL2-VL-VH(ScFv)-Fc(wt)).

本发明还涉及所述的双功能融合蛋白的如下应用：The present invention also relates to the following application of the bifunctional fusion protein:

(1)制备抗肿瘤药物；(1) Preparation of antitumor drugs;

(2)制备与免疫检查点抑制剂联用的抗肿瘤药物；(2) Preparation of anti-tumor drugs combined with immune checkpoint inhibitors;

(3)制备克服免疫检查点抑制剂耐受的抗肿瘤药物；(3) Preparation of anti-tumor drugs that overcome immune checkpoint inhibitor tolerance;

(4)制备与TKI拮抗剂联用的抗肿瘤药物；(4) preparation of antitumor drugs combined with TKI antagonists;

(5)制备克服TKI拮抗剂耐受的抗肿瘤药物。(5) Preparation of antitumor drugs that overcome TKI antagonist tolerance.

优选的，所述的免疫检查点抑制剂为PD-L1抗体；Preferably, the immune checkpoint inhibitor is PD-L1 antibody;

优选的，所述的TKI拮抗剂为小分子TKI拮抗剂；更优选的，所述的小分子TKI拮抗剂为阿法替尼或其结构类似物。Preferably, the TKI antagonist is a small molecule TKI antagonist; more preferably, the small molecule TKI antagonist is afatinib or a structural analog thereof.

本发明的有益效果在于：The beneficial effects of the present invention are:

(1)提出了IL2临床应用的瓶颈和解决方案，IL2/aOX40-hIgG1抗体在保留IL2对效应细胞活化的同时删除瘤内Treg细胞，克服了IL2使用过程中会导致Treg扩增的不利因素，这些结果为临床使用IL2提供了新思路；(1) The bottleneck and solution for the clinical application of IL2 are proposed. IL2/aOX40-hIgG1 antibody can delete intratumoral Treg cells while retaining the activation of effector cells by IL2, which overcomes the unfavorable factors that can lead to Treg expansion during the use of IL2. These results provide new ideas for the clinical use of IL2;

(2)对克服免疫检查点阻断疗法耐受和TKI化疗药使用后产生的抗性具有重要意义。(2) It is of great significance to overcome the resistance to immune checkpoint blockade therapy and the resistance generated after the use of TKI chemotherapy drugs.

附图说明Description of drawings

图1、IL2-Fc融合蛋白能部分控制肿瘤的生长，1A、IL2-Fc融合蛋白的分子结构，下部的二聚体为人IgG的Fc区二聚体，每个Fc单体N端偶联了一分子的野生型IL-2分子；1B、IL2-Fc融合蛋白对肿瘤生长的抑制效果。Figure 1. IL2-Fc fusion protein can partially control tumor growth. 1A. Molecular structure of IL2-Fc fusion protein. The lower dimer is the Fc region dimer of human IgG. The N-terminal of each Fc monomer is coupled to One molecule of wild-type IL-2 molecule; 1B, the inhibitory effect of IL2-Fc fusion protein on tumor growth.

图2、aOX40抗体刺激T细胞活化并发挥抗肿瘤功能，2A、aOX40抗体能够有效活化CD3 T细胞；2B、aOX40抗体能有效控制肿瘤生长。Figure 2. aOX40 antibody stimulates T cell activation and exerts anti-tumor function. 2A and aOX40 antibodies can effectively activate CD3 T cells; 2B and aOX40 antibodies can effectively control tumor growth.

图3、IL2-Fc和aOX40联合治疗未见明显的协同效果Figure 3. No obvious synergistic effect was found in the combined treatment of IL2-Fc and aOX40

图4、构建IL2/aOX40-Fc双特异性抗体，4A、IL2/aOX40-Fc双特异性抗体结构模式图；4B、SDS-PAGE分析构建的IL2/aOX40-Fc双特异性抗体；4C、IL2/aOX40-Fc异源二聚体形式，aOX40为Fab或scFv形式，抗体包含1个IL2分子；4D、IL2/aOX40-Fc，aOX40为scFv形式，在V区的N端或Fc的C端连接1个或2个IL2分子；4E、IL2/aOX40-Fc同源二聚体形式，aOX40为Fab形式，在轻链的N端或C端、在重链的N端或Fc的C端连接2个IL2分子；4F、IL2/aOX40-Fc异源二聚体形式，aOX40为Fab形式，在轻链的N端或C端、在重链的N端或Fc的C端连接1个IL2分子。Figure 4. Construction of IL2/aOX40-Fc bispecific antibody, 4A, structural pattern diagram of IL2/aOX40-Fc bispecific antibody; 4B, SDS-PAGE analysis of the constructed IL2/aOX40-Fc bispecific antibody; 4C, IL2 /aOX40-Fc heterodimer form, aOX40 is in Fab or scFv form, the antibody contains 1 IL2 molecule; 4D, IL2/aOX40-Fc, aOX40 is in scFv form, connected at the N-terminus of V region or the C-terminus of

Fc

1 or 2 IL2 molecules; 4E, IL2/aOX40-Fc homodimer form, aOX40 in Fab form, linked at N- or C-terminus of light chain, N-terminus of heavy chain or C-terminus of Fc 2 1 IL2 molecule; 4F, IL2/aOX40-Fc heterodimer form, aOX40 is in Fab form, one IL2 molecule is connected to the N-terminus or C-terminus of the light chain, the N-terminus of the heavy chain or the C-terminus of Fc.

图5、IL2/aOX40-Fc抗体具有协同治疗的效果，5A、体外CTLL2增殖实验；5B、IL2/aOX40 wt Fc融合蛋白的肿瘤抑制效果(MC38肿瘤模型)；5C、在IL2/aOX40-Fc治疗组re-challenge试验结果(MC38肿瘤模型)；5D、IL2/aOX40 wt Fc融合蛋白的肿瘤抑制效果(B16F10肿瘤模型)。Figure 5. IL2/aOX40-Fc antibody has synergistic therapeutic effect, 5A, in vitro CTLL2 proliferation assay; 5B, tumor inhibitory effect of IL2/aOX40 wt Fc fusion protein (MC38 tumor model); 5C, in IL2/aOX40-Fc treatment Group re-challenge test results (MC38 tumor model); tumor inhibitory effect of 5D, IL2/aOX40 wt Fc fusion protein (B16F10 tumor model).

图6、IL2/aOX40-Fc抗体治疗效果不依赖于NK细胞；6A、NK细胞删除性抗体注射后一天，流式分析小鼠外周血中NK细胞的比例；6B、不同治疗组MC38肿瘤的生长曲线。Figure 6. The therapeutic effect of IL2/aOX40-Fc antibody does not depend on NK cells; 6A, One day after NK cell-depleting antibody injection, the proportion of NK cells in the peripheral blood of mice was analyzed by flow cytometry; 6B, The growth of MC38 tumors in different treatment groups curve.

图7、IL2/aOX40 wt Fc功能依赖于CD8 T细胞；7A、注射200ug删除性抗体一天后，流式检测外周血中CD4 T细胞和CD8 T细胞删除效率；7B、不同治疗组肿瘤生长曲线。Figure 7. The function of IL2/aOX40 wt Fc depends on CD8 T cells; 7A, One day after injection of 200ug deletion antibody, the deletion efficiency of CD4 T cells and CD8 T cells in peripheral blood was detected by flow cytometry; 7B, The tumor growth curves of different treatment groups.

图8、瘤内T细胞对IL2/aOX40-Fc的治疗起关键作用；8A、MC38荷瘤小鼠腹腔注射20ug FTY720，2天后检测外周血CD3 T细胞；8B、不同治疗组肿瘤生长曲线。Figure 8. Intratumoral T cells play a key role in the treatment of IL2/aOX40-Fc; 8A, MC38 tumor-bearing mice were intraperitoneally injected with 20ug FTY720, and peripheral blood CD3 T cells were detected 2 days later; 8B, tumor growth curves in different treatment groups.

图9、原位肿瘤局部治疗诱导的免疫应答可以控制远端肿瘤生长；9A、不同治疗组右侧(给药侧)的肿瘤治疗效果；9B、不同治疗组左侧(非治疗侧)的肿瘤治疗效果。Figure 9. In situ tumor local treatment-induced immune response can control distal tumor growth; 9A, Tumor treatment effect on the right side (administration side) of different treatment groups; 9B, Tumors on the left side (non-treatment side) of different treatment groups treatment effect.

图10、CD25和OX40在瘤内Treg细胞上高表达；10A、流式分析肿瘤内CD4、CD8和Treg细胞上CD25、OX40的表达，比较瘤内、引流***和脾脏Treg细胞上CD25、OX40的表达；10B、不同细胞表达水平的MFI统计分析。Figure 10. CD25 and OX40 are highly expressed on intratumoral Treg cells; 10A. Flow cytometry analysis of the expressions of CD25 and OX40 on intratumoral CD4, CD8 and Treg cells, comparing the levels of CD25 and OX40 on intratumoral, draining lymph node and spleen Treg cells Expression; 10B, MFI statistical analysis of expression levels in different cells.

图11、IL2/aOX40-Fc治疗删除瘤内Treg提高CD8 T细胞与Treg的比例；11A、不同治疗组瘤内Treg细胞流式图；11B、不同治疗组瘤内Treg/CD4 T和CD8 T/Treg比例；11C、引流***和脾脏Treg/CD4 T比例。Figure 11. Deletion of intratumoral Treg by IL2/aOX40-Fc treatment increased the ratio of CD8 T cells to Treg; 11A, flow cytometry of intratumoral Treg cells in different treatment groups; 11B, intratumoral Treg/CD4 T and CD8 T/Treg cells in different treatment groups Treg ratio; 11C, draining lymph node and spleen Treg/CD4 T ratio.

图12、抗体功能的发挥部分依赖于Fc与FcγR的结合。Figure 12. The functioning of antibodies is partially dependent on the binding of Fc to FcγR.

图13、aPDL1抗体能够协同IL2/aOX40-Fc提高抗肿瘤效果。Figure 13. aPDL1 antibody can synergize with IL2/aOX40-Fc to improve the anti-tumor effect.

图14、IL2/aOX40-Fc能够克服Afatinib治疗的抗性，14A、14B、阿法替尼治疗过程中有更多的T细胞浸润到瘤内，但是Treg的比例没有改变；14C、阿法替尼和IL2/aOX40-Fc抗体联合治疗能够有效控制肿瘤的生长和复发。Figure 14. IL2/aOX40-Fc can overcome the resistance of afatinib treatment, 14A, 14B, more T cells infiltrated into the tumor during afatinib treatment, but the proportion of Treg did not change; 14C, afatinib Combination therapy with IL2/aOX40-Fc antibody can effectively control tumor growth and recurrence.

图15、IL2/aCTLA4 wt Fc融合蛋白增强肿瘤抑制效果(MC38肿瘤模型)。Figure 15. IL2/aCTLA4 wt Fc fusion protein enhances tumor suppressive effect (MC38 tumor model).

图16、IL2/a4-1BB-wt Fc双特异性抗体显著改善肿瘤治疗效果。Figure 16. The IL2/a4-1BB-wt Fc bispecific antibody significantly improved the tumor treatment effect.

具体实施方式Detailed ways

实验材料Experimental Materials

1、菌种和质粒1. Bacteria and plasmids

菌种：Top10 E.coli、DH5αE.coli感受态细胞(北京全式金生物技术有限公司)Bacteria: Top10 E.coli, DH5αE.coli competent cells (Beijing Quanshijin Biotechnology Co., Ltd.)

质粒：Plasmid:

pEE12.4-IgGκ-hIgG1，包含有小鼠IgGκ的信号肽和人IgG1的Fc序列，用于表达抗体。pEE12.4-IgGκ-hIgG1, which contains the signal peptide of mouse IgGκ and the Fc sequence of human IgG1, is used for antibody expression.

pEE12.4-IgGκ-hIgG1-Fc-hole和pEE12.4-IgGκ-hIgG1-Fc-knob用于表达异二聚体蛋白。pEE12.4-IgGκ-hIgG1-Fc-hole and pEE12.4-IgGκ-hIgG1-Fc-knob were used to express heterodimeric proteins.

001-OX40 VH-CH1-Fc-knob、002-OX40 VL-CL用于表达异二聚体蛋白的抗体部分；pEE12.4-Wt IL-2-Fc-hole、用于表达异二聚体蛋白的IL-2部分。001-OX40 VH-CH1-Fc-knob, 002-OX40 VL-CL for the expression of the antibody portion of the heterodimeric protein; pEE12.4-Wt IL-2-Fc-hole, for the expression of the heterodimeric protein part of the IL-2.

2、实验动物2. Experimental animals

野生型C57BL/6、BALB/c小鼠和BALB/c-nude小鼠购于中国北京维通利华实验动物心。除特殊说明外，所有实验使用的均为8-10周龄的雌性小鼠。小鼠均在无特定病原微生物(specific pathogen-free，SPF)的屏障环境中饲养。动物的饲养和实验操作遵从中国科学院生物物理研究所动物管理委员会的相关规定。Wild-type C57BL/6, BALB/c mice and BALB/c-nude mice were purchased from Weitong Lihua Laboratory Animal Center in Beijing, China. Unless otherwise stated, female mice aged 8-10 weeks were used in all experiments. Mice were raised in a specific pathogen-free (SPF) barrier environment. Animal breeding and experimental operations followed the relevant regulations of the Animal Management Committee of the Institute of Biophysics, Chinese Academy of Sciences.

3、细胞系3. Cell line

MC38为C57背景小鼠结直肠癌细胞系，MC38 is a C57 background mouse colorectal cancer cell line,

MC38-EGFR5是分别利用MC38感染表达人鼠嵌合的表皮生长因子(EGFR)的慢病毒并筛选得出的单克隆细胞系，MC38-EGFR5 are monoclonal cell lines obtained by infecting and screening lentivirus expressing human-mouse chimeric epidermal growth factor (EGFR) using MC38, respectively.

TUBO为BALB/c背景的小鼠乳腺癌细胞系，B16F10为C57背景小鼠黑色素瘤细胞系。TUBO is a mouse breast cancer cell line in BALB/c background, and B16F10 is a mouse melanoma cell line in C57 background.

上述这些细胞系均在DMEM完全培养基(含10％灭活胎牛血清，2mmol/l L-谷氨酰胺，0.1mmol/l非必需氨基酸，100U青霉素和100μg/ml链霉素)中培养。These cell lines were cultured in DMEM complete medium (containing 10% inactivated fetal bovine serum, 2 mmol/l L-glutamine, 0.1 mmol/l non-essential amino acids, 100 U penicillin and 100 μg/ml streptomycin).

TIB-210TM杂交瘤细胞系(ATCC)，用于表达CD8+T细胞的删除抗体(clone:2.43)。TIB-210TM hybridoma cell line (ATCC) for expression of CD8+ T cell deletion antibody (clone: 2.43).

TIB-207TM杂交瘤细胞系(ATCC)，用于表达CD4+T细胞的删除抗体(clone:GK1.5)。TIB-207TM hybridoma cell line (ATCC) for expression of CD4+ T cell deletion antibody (clone: GK1.5).

PK136杂交瘤细胞系用于生产NK细胞的删除抗体。The PK136 hybridoma cell line was used to produce NK cell deletion antibodies.

HB-197TM杂交瘤细胞系(ATCC)，用于表达阻断小鼠的FcγRII/III的抗体(clone:2.4G2)。HB-197TM hybridoma cell line (ATCC) for expression of an antibody that blocks FcγRII/III in mice (clone: 2.4G2).

FreeStyleTM 293F细胞系(Invitrogen)为悬浮细胞，源自于HEK293细胞株，培养在SMM293-TII或者CD OptiCHOTM培养基中，主要用于瞬时转染表达融合蛋白。FreeStyleTM 293F cell line (Invitrogen) is a suspension cell derived from HEK293 cell line, cultured in SMM293-TII or CD OptiCHOTM medium, and is mainly used for transient transfection and expression of fusion proteins.

CTLL-2细胞系为小鼠T细胞系，用于检测IL-2的生物活性CTLL-2 cell line is a mouse T cell line used to detect the biological activity of IL-2

上述细胞系在RPMI1640完全培养基(含10％灭活胎牛血清，2mmol/L L-谷氨酰胺，0.1mmol/L非必需氨基酸，100U青霉素和100μg/ml链霉素，100IU/ml重组IL2)中培养。The above cell lines were cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2mmol/L L-glutamine, 0.1mmol/L non-essential amino acids, 100U penicillin and 100μg/ml streptomycin, 100IU/ml recombinant IL2 ) in culture.

基因及引物的设计与合成Design and synthesis of genes and primers

人的野生型及突变型的IL-2基因序列如下SEQ ID NO.1所示。实验中所用引物均通过DNAMAN软件设计并由Invitrogen公司合成。The human wild-type and mutant IL-2 gene sequences are shown in SEQ ID NO.1 below. The primers used in the experiments were designed by DNAMAN software and synthesized by Invitrogen Company.

小鼠肿瘤接种及治疗Mouse tumor inoculation and treatment

(1)肿瘤接种及测量：(1) Tumor inoculation and measurement:

肿瘤模型的建立，establishment of tumor models,

5-7.5×10 ⁵个MC38、MC38-EGFR5单细胞悬于100μl PBS中，皮下接种于C57BL/6小鼠背部； 5-7.5×10 ⁵ MC38 and MC38-EGFR5 single cells were suspended in 100 μl PBS and subcutaneously inoculated on the back of C57BL/6 mice;

5-7.5×10 ⁵个TUBO单细胞悬于100μl PBS中,接种于BALB/c小鼠背部皮下。 5-7.5×10 ⁵ single TUBO cells were suspended in 100 μl PBS and inoculated subcutaneously on the back of BALB/c mice.

对肿瘤消退的小鼠进行同一种肿瘤细胞的re-challenge实验时，肿瘤细胞的接种数量为初始肿瘤造模时候的5倍，接种部位为小鼠背部另一侧皮下。每周监测两次肿瘤大小，使用游标卡尺测量肿瘤长径(a)、短径(b)和高(c)，小鼠肿瘤体积＝a×b×c/2。When re-challenge experiments with the same tumor cells were performed on mice with tumor regression, the number of tumor cells inoculated was 5 times that of the initial tumor modeling, and the inoculation site was subcutaneous on the other side of the back of the mice. The tumor size was monitored twice a week, and the long diameter (a), short diameter (b) and height (c) of the tumor were measured using a vernier caliper, mouse tumor volume=a×b×c/2.

(2)治疗：(2) Treatment:

抗体或者抗体融合蛋白采用腹腔注射方式，部分实验也采用肿瘤内给药的方式，具体给药剂量将在具体实验中叙述。The antibody or antibody fusion protein was injected intraperitoneally, and some experiments also used intratumoral administration, and the specific dosage will be described in the specific experiment.

单克隆抗体制备(小鼠腹水法)Monoclonal antibody preparation (mouse ascites method)

实验中用到的CD4+T细胞删除型抗体GK1.5、CD8+T细胞删除型抗体TIB210、NK细胞删除抗体PK136以及FcRII/III封闭抗体均来自相应的杂交瘤细胞，由本实验室生产、纯化。The CD4+ T cell-depleted antibody GK1.5, CD8+ T cell-depleted antibody TIB210, NK cell-deleted antibody PK136 and FcRII/III blocking antibody used in the experiment were all derived from the corresponding hybridoma cells, produced and purified by our laboratory. .

小鼠体内细胞删除Deletion of cells in mice

(1)CD4+T细胞、CD8+T细胞的删除：(1) Deletion of CD4+ T cells and CD8+ T cells:

IL-2或IL-2融合蛋白治疗前一天分别腹腔注射200μg GK1.5或者TIB210抗体用来删除CD4+T细胞、CD8+T细胞，之后每隔3天注射一次，依据治疗周期调整注射次数。通过流式检测删除效率。One day before IL-2 or IL-2 fusion protein treatment, 200 μg of GK1.5 or TIB210 antibody was injected intraperitoneally to delete CD4+ T cells and CD8+ T cells, and then injected every 3 days, and the number of injections was adjusted according to the treatment cycle. Detection of removal efficiency by streaming.

(2)NK细胞以及中性粒细胞的删除：(2) Deletion of NK cells and neutrophils:

IL-2或IL-2融合蛋白治疗前一天分别腹腔注射400μg PK136或者1A8抗体删除NK细胞或者中性粒细胞。流式检测删除效率。One day before IL-2 or IL-2 fusion protein treatment, 400 μg of PK136 or 1A8 antibody was injected intraperitoneally to delete NK cells or neutrophils, respectively. Streaming detection removal efficiency.

T细胞迁出阻断T cell emigration blockade

FTY720(购自Sigma公司)是一种免疫抑制剂，可以减少T细胞从淋巴器官迁出外周血液循环。在本发明中，小鼠肿瘤接种的不同时期进行FTY720阻断以改变肿瘤微环境。在对小鼠肿瘤治疗过程中进行阻断：肿瘤治疗前1天腹腔注射20μg FTY720，之后每隔一天进行腹腔注射10μg，阻断时间依据治疗周期而定，这样会造成在肿瘤治疗的过程中，没有新迁入肿瘤组织的T细胞。借助FTY720阻断方案，可以研究肿瘤组织中浸润的淋巴细胞的重要性。FTY720 (purchased from Sigma) is an immunosuppressive agent that reduces the migration of T cells from lymphoid organs out of the peripheral blood circulation. In the present invention, FTY720 blockade was performed at different periods of tumor inoculation in mice to alter the tumor microenvironment. Blocking during tumor treatment in mice: intraperitoneal injection of 20 μg FTY720 1 day before tumor treatment, followed by intraperitoneal injection of 10 μg every other day. There are no new T cells that have colonized the tumor tissue. With the help of the FTY720 blockade protocol, the importance of infiltrating lymphocytes in tumor tissue can be investigated.

如无特别说明，以下实施例1-5中的IL2/aOX40-Fc双特异性抗体的分子结构都为图4A所示的分子结构，即一分子IL2-Fc和一分子aOX40(Fab)-Fc组成的异源二聚体。Unless otherwise specified, the molecular structures of the IL2/aOX40-Fc bispecific antibodies in Examples 1-5 below are all the molecular structures shown in FIG. 4A , that is, a molecule of IL2-Fc and a molecule of aOX40(Fab)-Fc composed of heterodimers.

实施例1、IL2/aOX40-Fc双特异性抗体比单独治疗效果显著改善Example 1. IL2/aOX40-Fc bispecific antibody significantly improved the effect of treatment alone

1、IL2-Fc融合蛋白的治疗能部分控制肿瘤1. Treatment with IL2-Fc fusion protein can partially control tumors

单独的游离free IL2半衰期短且需要多次给药，为了延长IL2的半衰期我们设计了IL2-Fc融合蛋白(结构示意图见图1A)，并在MC38肿瘤模型中验证融合蛋白的功能。与未治疗的对照组相比IL2-Fc能够有效控制肿瘤的生长，但是在肿瘤较大时单独的IL2-Fc治疗未能完全清除肿瘤(图1B)。C57BL/6小鼠皮下接种5 ×10 ⁵MC38肿瘤细胞，荷瘤小鼠在D12天开始分组治疗(n＝5/组)：腹腔注射(ip)9ug IL2-Fc融合蛋白，每三天治疗一次，共治疗三次。 Free free IL2 alone has a short half-life and requires multiple administrations. In order to prolong the half-life of IL2, we designed an IL2-Fc fusion protein (see Figure 1A for a schematic diagram of the structure), and verified the function of the fusion protein in the MC38 tumor model. IL2-Fc was able to effectively control tumor growth compared to untreated controls, but IL2-Fc treatment alone failed to completely eliminate tumors when tumors were larger (Fig. 1B). C57BL/6 mice were subcutaneously inoculated with 5 × 10 ⁵ MC38 tumor cells, and the tumor-bearing mice were treated in groups on D12 (n=5/group): intraperitoneal injection (ip) of 9ug IL2-Fc fusion protein, once every three days , a total of three treatments.

结果显示，单独给予IL2-Fc融合蛋白治疗并不能持续抑制肿瘤的生长。The results showed that IL2-Fc fusion protein treatment alone did not sustainably inhibit tumor growth.

2、anti-OX40抗体促进T细胞活化并发挥抗肿瘤功能2. Anti-OX40 antibody promotes T cell activation and exerts anti-tumor function

单独IL2-Fc融合蛋白的治疗只能部分控制肿瘤的生长，为了进一步提高治疗的效果我们采取联合治疗的策略。OX40分子只在Treg细胞和活化的T细胞上表达而在

T细胞上不表达，这样靶向OX40的抗体具有较高的特异性；而且OX40在Treg细胞和效应细胞上的表达丰度不同，也为相同靶点对不同细胞产生不同效应提供了理论基础。为了验证OX40信号通路能否活化T细胞并发挥抗肿瘤作用，我们进行了脾脏

T细胞体外刺激实验。具体方法为：分离小鼠的脾脏细胞并按3×10 ⁵细胞/孔铺96孔板，分别用aCD3和aCD28或者aCD3和aOX40抗体刺激72h后流式分析CD3 T细胞上CD69的表达水平，aCD3、aCD28抗体浓度为1ug/ml，aOX40抗体浓度为2ug/ml。 The treatment of IL2-Fc fusion protein alone can only partially control the growth of the tumor. In order to further improve the effect of the treatment, we adopt a combination therapy strategy. OX40 molecule is only expressed on Treg cells and activated T cells

It is not expressed on T cells, so the antibody targeting OX40 has higher specificity; and the expression abundance of OX40 on Treg cells and effector cells is different, which also provides a theoretical basis for the same target to produce different effects on different cells. To verify whether the OX40 signaling pathway can activate T cells and exert anti-tumor effects, we performed spleen

T cell stimulation experiments in vitro. The specific method is as follows: Isolate mouse spleen cells and spread 3×10 ⁵ cells/well in 96-well plates. After stimulation with aCD3 and aCD28 or aCD3 and aOX40 antibodies for 72 h, flow cytometry analysis of the expression level of CD69 on CD3 T cells, aCD3 , aCD28 antibody concentration is 1ug/ml, aOX40 antibody concentration is 2ug/ml.

结果显示，相比较aCD3和aCD28对T细胞的共刺激效应，aOX40抗体和aCD3联合使用发挥了和aCD28相类似的刺激作用，能够有效活化CD3 T细胞(图2A)。The results showed that compared with the co-stimulatory effects of aCD3 and aCD28 on T cells, the combination of aOX40 antibody and aCD3 exerted a similar stimulatory effect as aCD28, and could effectively activate CD3 T cells (Figure 2A).

我们进一步在小鼠的抗肿瘤实验中验证anti-OX40的功能，具体方法为：野生型C57BL/6小鼠皮下接种5×10 ⁵MC38肿瘤细胞，肿瘤生长到100-150mm ³开始分组治疗(n＝5/组)：腹腔注射(ip)16ug aOX40抗体，三天治疗一次共治疗三次。 We further verified the function of anti-OX40 in anti-tumor experiments in mice. The specific method was as follows: wild-type C57BL/6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and the tumors grew to 100-150 mm ³ to start group therapy (n =5/group): intraperitoneal injection (ip) of 16ug of aOX40 antibody, once every three days for a total of three treatments.

结果显示，与未治疗组相比，腹腔注射(ip)16ug aOX40抗体能有效控制肿瘤生长(图2B)。The results showed that intraperitoneal (ip) injection of 16ug aOX40 antibody effectively controlled tumor growth compared with the untreated group (Fig. 2B).

3、IL2-Fc融合蛋白和aOX40联合治疗并未明显提高肿瘤的治疗效果3. Combination therapy of IL2-Fc fusion protein and aOX40 did not significantly improve the therapeutic effect of tumor

IL2-Fc单独使用会扩增Treg，而aOX40抗体能够删除瘤内Treg细胞，为了验证IL2-Fc和aOX40抗体联合使用是否能够在活化效应T细胞同时删除扩增的Treg，发挥协同治疗的效果。我们在MC38荷瘤小鼠上同时给予两种蛋白治疗，方法为：C57BL6小鼠皮下接种5×10 ⁵MC38肿瘤细胞，肿瘤生长到D14开始治疗，单独的治疗组分别腹腔注射(ip)6ug IL2-Fc或者9ug aOX40抗体，联合治疗组同时腹腔注射两种抗体；每三天治疗一次共治疗三次。 IL2-Fc alone can expand Treg, while aOX40 antibody can delete intratumoral Treg cells. In order to verify whether the combined use of IL2-Fc and aOX40 antibody can activate effector T cells and delete expanded Treg, it can play a synergistic therapeutic effect. We simultaneously administered two protein treatments to MC38 tumor-bearing mice. The method was as follows: C57BL6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and the tumor grew to D14 to start treatment, and the individual treatment groups were intraperitoneally injected (ip) with 6ug IL2 -Fc or 9ug aOX40 antibody, the combined treatment group was injected with both antibodies at the same time; once every three days for a total of three treatments.

结果显示，与单独的aOX40抗体或者IL2-Fc治疗相比，联合治疗并未能进一步提高IL2-Fc的治疗效果(图3)。The results showed that the combination therapy did not further improve the therapeutic effect of IL2-Fc compared with aOX40 antibody or IL2-Fc treatment alone (Figure 3).

4、IL2/aOX40-Fc双特异性抗体具有协同治疗的效果4. IL2/aOX40-Fc bispecific antibody has synergistic therapeutic effect

4.1、双特异性抗体的构建4.1. Construction of bispecific antibodies

由上述实验结果分析发现，简单的混合性联合治疗并不具有明显的协同治疗效果，甚至没有叠加效果，提示我们两种抗体单独结合并不能同时发挥出既活化效应细胞又删除Treg的效果。据此，为了降低IL2结合、扩增Treg细胞，我们构建了两种结构一致的双特异性抗体(IL2/aOX40 wt Fc和IL2/aOX40 no ADCC Fc)，每个抗体分子仅含有一个IL2分子(融合蛋白结构见图4A)，该分子中，Fc二聚体为人IgG的Fc二聚体或删除ADCC效应功能的Fc二聚体，一条Fc单体与WT型IL2蛋白连接，另一条Fc单体与aOX40抗体的Fab段连接。构建完成的融合蛋白质粒转染293F细胞，七天后收细胞上清。离心上清后用protein A柱子纯化蛋白，纯化后的抗体经蛋白胶鉴定。经SDS-PAGE分析蛋白分子组成，证明双特异性分子能够同时完好表达aOX40抗体的轻链、重链和IL2分子(图4B)。From the analysis of the above experimental results, it is found that the simple mixed combination therapy does not have obvious synergistic therapeutic effect, or even has no additive effect, suggesting that the combination of the two antibodies alone cannot exert the effect of activating effector cells and deleting Treg at the same time. Accordingly, in order to reduce IL2 binding and expand Treg cells, we constructed two structurally identical bispecific antibodies (IL2/aOX40 wt Fc and IL2/aOX40 no ADCC Fc), each containing only one IL2 molecule ( The structure of the fusion protein is shown in Figure 4A). In this molecule, the Fc dimer is the Fc dimer of human IgG or the Fc dimer with the ADCC effector function deleted. One Fc monomer is connected to the WT IL2 protein, and the other Fc monomer Linked to the Fab fragment of aOX40 antibody. The constructed fusion protein particles were transfected into 293F cells, and the cell supernatant was collected seven days later. After centrifuging the supernatant, the protein was purified by protein A column, and the purified antibody was identified by protein gel. The molecular composition of the protein was analyzed by SDS-PAGE, and it was proved that the bispecific molecule could fully express the light chain, heavy chain and IL2 molecule of aOX40 antibody simultaneously (Fig. 4B).

我们同时设计和构建了包含IL2和aOX40不同形式的抗体：We simultaneously designed and constructed antibodies containing different forms of IL2 and aOX40:

图4C示意的结构为异源二聚体，抗体包含一个IL2分子，aOX40(抗OX40抗体)为Fab或scFv形式，Fc二聚体为人IgG的Fc单体和删除ADCC效应功能的人IgG的Fc单体形成的异源二聚体。The structure shown in Figure 4C is a heterodimer, the antibody contains an IL2 molecule, aOX40 (anti-OX40 antibody) is in the form of Fab or scFv, and the Fc dimer is the Fc monomer of human IgG and the Fc of human IgG with the ADCC effector function deleted. Heterodimers formed from monomers.

图4D示意的结构中，aOX40(抗OX40抗体)为scFv形式，在V区的N端或Fc的C端连接一个或两个IL2分子，Fc二聚体为野生型人IgG的Fc同源二聚体，或人IgG的Fc单体和删除ADCC效应功能的人IgG的Fc单体形成的异源二聚体；In the structure shown in Figure 4D, aOX40 (anti-OX40 antibody) is in the form of scFv, one or two IL2 molecules are connected to the N-terminus of the V region or the C-terminus of the Fc, and the Fc dimer is the Fc homologous two of wild-type human IgG. A polymer, or a heterodimer formed by the Fc monomer of human IgG and the Fc monomer of human IgG with ADCC effector function deleted;

图4 E-F示意的结构中，aOX40(抗OX40抗体)为Fab形式，在抗体轻链的N端或C端、在抗体重链的N端或Fc的C端连接一个或两个IL2分子，Fc二聚体为野生型人IgG的Fc同源二聚体，或人IgG的Fc单体和删除ADCC效应功能的人IgG的Fc单体形成的异源二聚体。In the structure shown in Figure 4 E-F, aOX40 (anti-OX40 antibody) is in the form of Fab, and one or two IL2 molecules are connected to the N-terminus or C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain or the C-terminus of the Fc. The dimer is an Fc homodimer of wild-type human IgG, or a heterodimer formed by an Fc monomer of human IgG and an Fc monomer of human IgG with the ADCC effector function deleted.

4.2、验证双特异性抗体的功能，4.2. Verify the function of bispecific antibodies,

(1)体外CTLL2增殖实验：(1) In vitro CTLL2 proliferation experiment:

在CTLL2细胞培养基中加入不同浓度的IL2-Fc、IL2/aOX40 wt Fc和IL2/aOX40 no ADCC Fc蛋白(图4A所示结构)，72h后通过CCK8试剂盒检测CTLL2细胞在不同蛋白浓度下的增值水平，结果显示IL2/aOX40-Fc抗体和IL2-Fc分子一样能够有效的扩增CTLL2细胞，说明构建的抗体分子保留了IL2活性(图5A)。Different concentrations of IL2-Fc, IL2/aOX40 wt Fc and IL2/aOX40 no ADCC Fc proteins (structure shown in Figure 4A) were added to the CTLL2 cell culture medium. The results showed that the IL2/aOX40-Fc antibody could effectively expand CTLL2 cells as well as the IL2-Fc molecule, indicating that the constructed antibody molecule retained IL2 activity (Fig. 5A).

(2)体内小鼠荷瘤实验：(2) In vivo mouse tumor-bearing experiment:

在C57BL6小鼠皮下接种5×10 ⁵MC38肿瘤细胞，在接种肿瘤后D14开始治疗；分别通过腹腔注射9ug IL2-Fc、16ug aOX40抗体、与aOX40抗体等摩尔数的IL2-Fc+aOX40抗体或IL2/aOX40 wt Fc蛋白。在D14、D17、D20天共治疗三次，每周两次测量肿瘤大小；结果显示，与单独的aOX40抗体、IL2-Fc以及两者混合治疗相比，IL2/aOX40-wtFc双特异抗体具有更好的治疗效果(图5B)。 C57BL6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and treatment was started at D14 after tumor inoculation; 9ug IL2-Fc, 16ug aOX40 antibody, IL2-Fc + aOX40 antibody or IL2 in equimolar numbers of aOX40 antibody were injected intraperitoneally, respectively /aOX40 wt Fc protein. Three treatments were performed on days D14, D17, and D20, and tumor size was measured twice a week; the results showed that the IL2/aOX40-wtFc bispecific antibody was better than aOX40 antibody alone, IL2-Fc, and the combination of both. treatment effect (Figure 5B).

除了MC38模型外，在B16F10荷瘤小鼠模型上也进行同样的实验，在C57BL6小鼠皮下接种5×10 ⁵B16F10肿瘤细胞，在接种肿瘤后D8开始治疗；通过腹腔注射15ug IL2/aOX40 wt Fc蛋白；在D8、D11和D14天共治疗三次，结果显示，IL2/aOX40 wt Fc也能发挥显著的肿瘤控制作用(图5D)。 In addition to the MC38 model, the same experiment was performed on the B16F10 tumor-bearing mouse model. C57BL6 mice were subcutaneously inoculated with 5×10 ⁵ B16F10 tumor cells, and treatment was started at D8 after tumor inoculation; 15ug IL2/aOX40 wt Fc was injected intraperitoneally protein; treated three times on days D8, D11 and D14, the results showed that IL2/aOX40 wt Fc also exerted a significant tumor control effect (Fig. 5D).

进一步的，在IL2/aOX40-Fc治疗组，肿瘤清除后两个月进行肿瘤re-challenge实验。在IL2/aOX40 wt Fc组，治疗完全清除肿瘤的小鼠两个月后进行肿瘤re-challenge试验，皮下接种五倍剂量的(2.5×10 ⁶个)MC38肿瘤细胞，re-challenge只接种肿瘤细胞，接种后各组均不再给药治疗。 Further, in the IL2/aOX40-Fc treatment group, tumor re-challenge experiments were performed two months after tumor clearance. In the IL2/aOX40 wt Fc group, mice that had completely cleared tumors were treated with tumor re-challenge for two months. Five times the dose (2.5×10 ⁶ cells) of MC38 tumor cells were subcutaneously inoculated in the re-challenge. Only tumor cells were inoculated for re-challenge. , after inoculation, each group was no longer given any treatment.

结果显示，与对照组相比，再次接种五倍剂量的MC38细胞并不会引起肿瘤的生长，说明双特异性抗体治疗诱导小鼠产生记忆性免疫细胞(图5C)。The results showed that re-inoculation with five times the dose of MC38 cells did not cause tumor growth compared with the control group, indicating that the bispecific antibody treatment induced the production of memory immune cells in the mice (Fig. 5C).

实施例2、IL2/aOX40-Fc能够激活CD8 T细胞Example 2. IL2/aOX40-Fc can activate CD8 T cells

1、IL2/aOX40-Fc双特异抗体治疗效果不依赖于NK细胞1. The therapeutic effect of IL2/aOX40-Fc bispecific antibody does not depend on NK cells

由于CD25(IL-2受体α)和OX40主要表达在活化的效应T细胞和NK细胞上，为了确定IL2/aOX40-Fc抗体的治疗主要依赖于哪一群免疫细胞，我们分别进行了不同细胞群的删除实验。Since CD25 (IL-2 receptor alpha) and OX40 are mainly expressed on activated effector T cells and NK cells, in order to determine which population of immune cells the treatment of IL2/aOX40-Fc antibody mainly depends on, we performed different cell populations. delete experiment.

C57BL6小鼠皮下接种5×10 ⁵MC38肿瘤细胞，在第12、15、18天腹腔注射25ug IL2/aOX40 wt Fc融合蛋白。在治疗开始前一天腹腔注射400ug NK细胞删除性抗体PK136(本实验室制备)，每4天一次，共注射3次。 C57BL6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and 25ug IL2/aOX40 wt Fc fusion protein was injected intraperitoneally on days 12, 15 and 18. 400ug of NK cell-depleting antibody PK136 (prepared in our laboratory) was intraperitoneally injected one day before the treatment, once every 4 days, for a total of 3 injections.

图6A显示在删除NK细胞1天后，外周血NK细胞的变化情况。在小鼠肿瘤实验中，删除NK细胞后IL2/aOX40-Fc抗体仍然具有治疗效果，说明NK细胞不是抗体发挥治疗效果的主要效应细胞(图6B)。Figure 6A shows the changes of peripheral blood NK cells 1 day after deletion of NK cells. In mouse tumor experiments, the IL2/aOX40-Fc antibody still had a therapeutic effect after NK cell deletion, indicating that NK cells were not the main effector cells for the antibody to exert its therapeutic effect (Fig. 6B).

2、IL2/aOX40-Fc抗体治疗效果依赖于CD8 T细胞2. The therapeutic effect of IL2/aOX40-Fc antibody depends on CD8 T cells

我们进一步验证T细胞在抗体治疗过程中发挥的作用。We further validated the role of T cells in the process of antibody therapy.

C57小鼠背部皮下接种5×10 ⁵MC38肿瘤细胞后第12天开始治疗，腹腔注射25ug IL2/aOX40 wt Fc蛋白(肿瘤接种后分别在第12、15、18天给药)。在治疗的同时腹腔注射200ug CD4 T细胞删除性抗体(克隆号：GK1.5，本实验室制备)、200ug CD8 T细胞删除性抗体(克隆号：TIB210，本实验室制备)或者同时注射两种删除性抗体，共注射三次。 C57 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells on the back of the 12th day, and were intraperitoneally injected with 25ug IL2/aOX40 wt Fc protein (administered on the 12th, 15th, and 18th days after tumor inoculation, respectively). At the same time of treatment, 200ug CD4 T cell-depleting antibody (clone number: GK1.5, prepared in our laboratory), 200ug CD8 T cell-depleting antibody (clone number: TIB210, prepared in our laboratory) or both were injected intraperitoneally. Deleting antibodies were injected three times in total.

CD4 T和CD8 T细胞的删除实验表明，删除CD4 T细胞后抗体治疗效果无显著下降，但在删除CD8 T细胞后，治疗效果有明显的降低；同时删除CD4 T和CD8 T细胞后，抗体治疗效果也完全消失。说明IL2/aOX40-Fc抗体的治疗效果依赖于T细胞，而且主要是CD8 T细胞(图7B)。The deletion experiment of CD4 T and CD8 T cells showed that the effect of antibody treatment did not decrease significantly after CD4 T cell deletion, but the therapeutic effect was significantly reduced after CD8 T cell deletion; after both CD4 T and CD8 T cell deletion, antibody treatment The effect is also completely gone. This indicated that the therapeutic effect of IL2/aOX40-Fc antibody was dependent on T cells, and mainly CD8 T cells (Fig. 7B).

3、IL2/aOX40-Fc双特异型抗体的功能依赖于瘤内的T细胞3. The function of IL2/aOX40-Fc bispecific antibody depends on intratumoral T cells

为了进一步检测治疗过程中是瘤内浸润的T细胞发挥主要作用还是外周迁移的T细胞发挥主要作用，我们进行了FTY720阻断实验。FTY720是临床上可以抑制器官移植过程中免疫排斥反应的一种药物，抑制T细胞由淋巴器官向外周血的迁移[19]。To further examine whether intratumorally infiltrating T cells or peripherally migrating T cells play a major role during treatment, we performed FTY720 blockade experiments. FTY720 is a clinical drug that can inhibit immune rejection during organ transplantation, and inhibit the migration of T cells from lymphoid organs to peripheral blood [19].

单独的IL2/aOX40 wt Fc抗体治疗可以清除肿瘤，在治疗的同时进行FTY720的阻断，实验方案如下：IL2/aOX40 wt Fc antibody treatment alone can clear the tumor, and block FTY720 at the same time of treatment. The experimental protocol is as follows:

C57BL6小鼠背部皮下接种4×10 ⁵MC38肿瘤细胞，在肿瘤接种后第7、10和13天，腹腔注射15ug IL2/aOX40-Fc抗体。在FTY720处理组，在肿瘤接种后第6天腹腔注射25ug FTY720，并继续在第8，10和12天分别注射10ug。 C57BL6 mice were subcutaneously inoculated with 4×10 ⁵ MC38 tumor cells on the back, and 15ug IL2/aOX40-Fc antibody was injected intraperitoneally on days 7, 10 and 13 after tumor inoculation. In the FTY720-treated group, 25ug of FTY720 was injected intraperitoneally on the 6th day after tumor inoculation, and 10ug was injected on the 8th, 10th and 12th days, respectively.

结果显示，对外周淋巴细胞向肿瘤迁移的抑制并不影响双特异抗体治疗效果(图8B)，表明IL2/aOX40-Fc抗体发挥抗肿瘤作用主要依赖于浸润到瘤内的效应T细胞。The results showed that the inhibition of the migration of peripheral lymphocytes to the tumor did not affect the therapeutic effect of the bispecific antibody (Fig. 8B), indicating that the anti-tumor effect of the IL2/aOX40-Fc antibody mainly depends on the effector T cells infiltrating into the tumor.

4、IL2/aOX40-Fc的治疗对远端转移型肿瘤的保护作用4. The protective effect of IL2/aOX40-Fc treatment on distant metastatic tumors

为了验证原位的肿瘤治疗产生的免疫保护是否对其它转移位点的肿瘤也具有抑制作用，我们进行了小鼠双肿瘤模型实验。我们同时在小鼠皮下左右两侧分别接种肿瘤细胞并只在其中一侧进行原位治疗，观察远端的肿瘤是否也能得到控制。实验方案如下：In order to verify whether the immune protection produced by in situ tumor treatment also has inhibitory effects on tumors at other metastatic sites, we performed a mouse double tumor model experiment. At the same time, we inoculated tumor cells on the left and right sides of the mouse subcutaneously and performed in situ treatment on only one side to observe whether the distal tumor could also be controlled. The experimental scheme is as follows:

C57BL6小鼠在背部的左右两侧分别接种2×10 ⁵MC38和4×10 ⁵MC38肿瘤细胞(在背部右侧接种4×10 ⁵MC38肿瘤细胞，在左侧接种2×10 ⁵MC38肿瘤细胞)。在第七天右侧的肿瘤长到50mm ³左右，左侧的肿瘤在30mm ³左右开始治疗： C57BL6 mice were inoculated with 2×10 ⁵ MC38 and 4×10 ⁵ MC38 tumor cells on the left and right sides of the back (4×10 ⁵ MC38 tumor cells were inoculated on the right side of the back and 2×10 ⁵ MC38 tumor cells were inoculated on the left side) . On the seventh day the tumor on the right side grew to around ^50mm3 and the tumor on the left side started treatment at around ^30mm3 :

治疗组1，在右侧肿瘤的瘤内注射7.5ug IL2/aOX40 wt Fc抗体(治疗抗体在第7、10、13天共治疗三次)，左侧肿瘤不治疗；In treatment group 1, 7.5ug IL2/aOX40 wt Fc antibody was injected into the tumor on the right side (the therapeutic antibody was treated three times on days 7, 10, and 13), and the tumor on the left side was not treated;

治疗组2，在右侧肿瘤的瘤内注射7.5ug IL2/aOX40 wt Fc抗体，同时在右侧肿瘤瘤内注射20ug TIB210抗体，删除瘤内CD8 T细胞。治疗和删除抗体在第7、10、13天共治疗三次，左侧肿瘤不治疗。In treatment group 2, 7.5ug of IL2/aOX40 wt Fc antibody was injected into the right tumor, and 20ug of TIB210 antibody was injected into the right tumor to delete intratumoral CD8 T cells. Treatment and deletion antibodies were treated three times on days 7, 10, and 13, and left tumors were left untreated.

结果如图9所示，The result is shown in Figure 9,

(1)仅对右侧肿瘤进行瘤内注射的原位治疗，不仅能够清除原位的肿瘤而且能够控制对侧远端未治疗的肿瘤。(1) In situ treatment with intratumoral injection only on the right tumor can not only remove the in situ tumor but also control the contralateral distal untreated tumor.

(2)同时为了验证远端肿瘤的控制是否依赖于治疗侧原位肿瘤的CD8 T细胞，在治疗的同时我们删除原位肿瘤的CD8 T细胞。在删除CD8 T细胞后，原位和远端肿瘤治疗效果都消失了，说明原位肿瘤内的CD8 T细胞对控制远端肿瘤发挥重要的作用。(2) At the same time, in order to verify whether the control of the distal tumor depends on the CD8 T cells of the orthotopic tumor on the treatment side, we deleted the CD8 T cells of the orthotopic tumor at the same time of treatment. After deletion of CD8 T cells, the therapeutic effect of both orthotopic and distal tumors disappeared, indicating that CD8 T cells in orthotopic tumors play an important role in controlling distal tumors.

实施例3、IL2/aOX40-Fc能够删除瘤内Treg并提高CD8/Treg细胞比例Example 3. IL2/aOX40-Fc can delete intratumoral Treg and increase the ratio of CD8/Treg cells

1、瘤内Treg细胞高表达CD25和OX40分子1. Intratumoral Treg cells highly express CD25 and OX40 molecules

为了进一步研究IL2/aOX40-Fc抗体的抗肿瘤机制，我们分析了荷瘤小鼠T细胞上CD25和OX40分子的表达水平以分析IL2/aOX40-Fc抗体主要结合到哪一群细胞上发挥功能。在接种MC38肿瘤后，第12天我们分别分析小鼠肿瘤内、引流***和脾脏T细胞上IL2受体CD25和OX40的表达：具体方案：小鼠接种5×10 ⁵MC38肿瘤细胞，在第12天分别取荷瘤小鼠的肿瘤组织、脾脏和引流***(dLN)，流式检测不同细胞上CD25和OX40表达。 In order to further study the anti-tumor mechanism of IL2/aOX40-Fc antibody, we analyzed the expression levels of CD25 and OX40 molecules on T cells of tumor-bearing mice to analyze which group of cells the IL2/aOX40-Fc antibody mainly binds to for function. After inoculation with MC38 tumors, we analyzed the expression of IL2 receptors CD25 and OX40 on T cells in the tumor, draining lymph nodes, and spleen of mice on the 12th day: specific protocol: mice were inoculated with 5×10 ⁵ MC38 tumor cells, and on the 12th day The tumor tissue, spleen and draining lymph node (dLN) of the tumor-bearing mice were collected on the 1st day respectively, and the expressions of CD25 and OX40 on different cells were detected by flow cytometry.

发现在荷瘤小鼠中，CD25和OX40均在Treg细胞上高表达，而且瘤内Treg比外周引流***和脾脏Treg表达更高水平CD25和OX40。非Treg的CD4 T和CD8 T的表达水平比Treg细胞低，瘤内的CD8 T细胞的CD25和OX40表达水平最低(图10A、B)。It was found that in tumor-bearing mice, both CD25 and OX40 were highly expressed on Treg cells, and intratumoral Treg expressed higher levels of CD25 and OX40 than peripheral draining lymph node and spleen Treg. Non-Treg CD4 T and CD8 T expression levels were lower than Treg cells, and intratumoral CD8 T cells had the lowest expression levels of CD25 and OX40 (Fig. 10A, B).

2、IL2/aOX40-Fc抗体删除瘤内Treg细胞，提高CD8/Treg细胞比例2. IL2/aOX40-Fc antibody deletes intratumoral Treg cells and increases the ratio of CD8/Treg cells

由于CD25和OX40高表达在瘤内的Treg细胞上，我们推测IL2/aOX40-Fc抗体腹腔注射后可能更多的结合到Treg上。在MC38荷瘤小鼠在接种肿瘤后第12天开始治疗，腹腔注射25ug IL2/aOX40-Fc抗体，在第15天取肿瘤组织、引流***和脾脏，流式分析瘤内Treg细胞的比例、CD8 T/Treg。通过流式实验分析，我们发现相比于IL2-Fc和aOX40的单独治疗，IL2/aOX40-Fc能够更有效的删除瘤内的Treg细胞，并提高CD8 T/Treg比例而具有最好的治疗效果(图11A，B)。相比较于对瘤内Treg的删除，IL2/aOX40-Fc并不改变外周肿瘤引流***和脾脏中Treg/CD4 T比例，说明IL2/aOX40-Fc治疗不会打破外周的免疫耐受具有更好的安全性(图11C)。Since CD25 and OX40 were highly expressed on intratumoral Treg cells, we speculated that IL2/aOX40-Fc antibody might bind more to Treg after intraperitoneal injection. MC38 tumor-bearing mice were treated on the 12th day after tumor inoculation, and 25ug IL2/aOX40-Fc antibody was injected intraperitoneally. On the 15th day, tumor tissue, draining lymph nodes and spleen were collected, and the proportion of Treg cells in the tumor, CD8 T/Treg. Through flow analysis, we found that compared with IL2-Fc and aOX40 alone, IL2/aOX40-Fc can more effectively delete intratumoral Treg cells and increase the ratio of CD8 T/Treg to have the best therapeutic effect (FIG. 11A,B). Compared with the deletion of intratumoral Treg, IL2/aOX40-Fc did not change the Treg/CD4 T ratio in peripheral tumor-draining lymph nodes and spleen, indicating that IL2/aOX40-Fc treatment does not break peripheral immune tolerance and has a better effect. Safety (FIG. 11C).

3、IL2/aOX40-Fc抗体治疗效果依赖于Fc与FcγR的结合3. The therapeutic effect of IL2/aOX40-Fc antibody depends on the binding of Fc to FcγR

通过对MC38荷瘤小鼠瘤内淋巴细胞的分析，我们发现IL2/aOX40-Fc抗体具有删除Treg的作用，为了验证Treg删除是否是IL2/aOX40-Fc抗体发挥抗肿瘤作用的主要机制，我们通过对Fc区域点突变构建了IL2/aOX40-no ADCC Fc抗体，突变后的Fc不与FcγR受体结合而失去ADCC、ADCP介导的删除功能。使用MC38肿瘤模型，C57BL6小鼠接种MC38肿瘤细胞，接种后第13天开始治疗。腹腔注射15ug wt Fc或者no ADCC Fc抗体，每三天治疗一次，共治疗三次。By analyzing the intratumoral lymphocytes of MC38 tumor-bearing mice, we found that IL2/aOX40-Fc antibody has the effect of deleting Treg. The IL2/aOX40-no ADCC Fc antibody was constructed by point mutation of the Fc region. The mutated Fc did not bind to the FcγR receptor and lost the deletion function mediated by ADCC and ADCP. Using the MC38 tumor model, C57BL6 mice were inoculated with MC38 tumor cells, and treatment was initiated on day 13 after inoculation. 15ug wt Fc or no ADCC Fc antibody was injected intraperitoneally, once every three days, for a total of three treatments.

结果显示，接种肿瘤后，对比wt Fc与no ADCC Fc的治疗效果，发现突变的Fc仅部分降低了IL2/aOX40抗体的治疗效果(图12)。IL2/aOX40-no ADCC Fc抗体治疗组中，抗体仍然保留部分抗肿瘤作用，说明IL2/aOX40-Fc抗体功能的发挥不仅仅依赖对Treg细胞进行删除，还可以通过与效应T细胞的结合发挥治疗作用。The results showed that after tumor inoculation, comparing the therapeutic effect of wt Fc and no ADCC Fc, it was found that the mutated Fc only partially reduced the therapeutic effect of IL2/aOX40 antibody (Figure 12). In the IL2/aOX40-no ADCC Fc antibody treatment group, the antibody still retained part of its anti-tumor effect, indicating that the function of IL2/aOX40-Fc antibody does not only depend on the deletion of Treg cells, but also can be combined with effector T cells to play a therapeutic role effect.

实施例4、IL2/aOX40-Fc联合PDL1抗体治疗Example 4. IL2/aOX40-Fc combined with PDL1 antibody therapy

1、aPDL1能够协同IL2/aOX40-Fc抗体增强抗肿瘤效果1. aPDL1 can synergize with IL2/aOX40-Fc antibody to enhance anti-tumor effect

在MC38荷瘤小鼠肿瘤在200mm ³以下时，单独的IL2/aOX40-Fc抗体治疗具有很好的清除作用。但是当肿瘤更大时，单独的抗体治疗只能控制肿瘤生长而不能完全清除；为了更好的提高治疗效果，我们将双特异性抗体和免疫检查点抑制性抗体联合使用观察能否提高治疗效果。 IL2/aOX40-Fc antibody treatment alone had good clearance when tumors in MC38 tumor-bearing mice were below 200 mm ³ . However, when the tumor is larger, antibody therapy alone can only control the growth of the tumor and cannot completely remove it; in order to better improve the therapeutic effect, we will use a combination of bispecific antibodies and immune checkpoint inhibitory antibodies to observe whether the therapeutic effect can be improved. .

具体试验方案：Specific test plan:

方案一：C57BL6小鼠皮下接种MC38细胞并在第16天开始治疗，Scheme 1: C57BL6 mice were subcutaneously inoculated with MC38 cells and started treatment on day 16,

(1)单独的IL2/aOX40-Fc治疗组第16、19和22天腹腔注射25ug IL2/aOX40-Fc；(1) Intraperitoneal injection of 25ug IL2/aOX40-Fc on days 16, 19 and 22 of the IL2/aOX40-Fc treatment group alone;

(2)aOX40和aCTLA4联合组第16天腹腔注射250ug抗体两天后注射25ug IL2/aOX40-Fc(aOX40和aCTLA4抗体仅给药一次，25ug IL2/aOX40-Fc腹腔注射给药三次)；(2) The aOX40 and aCTLA4 combination group was injected intraperitoneally with 250ug of antibody on day 16 and then injected with 25ug of IL2/aOX40-Fc (aOX40 and aCTLA4 antibodies were administered only once, and 25ug of IL2/aOX40-Fc was administered by intraperitoneal injection three times);

(3)aPDL1联合治疗组在注射25ug IL2/aOX40-Fc同时注射250ug aPDL1抗体(16、19、22天)。(3) The aPDL1 combination treatment group was injected with 25ug IL2/aOX40-Fc and 250ug aPDL1 antibody (days 16, 19, and 22).

方案二：MC38荷瘤小鼠，第12、15和18天腹腔注射200ug aPDL1抗体，第14、17和20天腹腔注射15ug wt Fc或者no ADCC Fc的IL2/aOX40抗体。Scheme 2: MC38 tumor-bearing mice were injected intraperitoneally with 200ug aPDL1 antibody on days 12, 15 and 18, and 15ug wt Fc or no ADCC Fc IL2/aOX40 antibody on days 14, 17 and 20.

结果显示：The results show:

(1)在IL2/aOX40-Fc与aCTLA4或aOX40联合治疗的过程中，发现这两类抗体的联合使用并不能进一步提高IL2/aOX40-Fc的治疗效果(图13A)。(1) During the combined treatment of IL2/aOX40-Fc with aCTLA4 or aOX40, it was found that the combined use of these two types of antibodies could not further improve the therapeutic effect of IL2/aOX40-Fc (Fig. 13A).

(2)在和aPDL1的联合治疗过程中发现，两者的联合使用具有协同作用效果(图13A)。(2) In the course of combined treatment with aPDL1, it was found that the combined use of the two has a synergistic effect (Fig. 13A).

(3)为了进一步分析aPDL1与IL2/aOX40-Fc抗体的作用机制，我们分别使用wt Fc或者no ADCC Fc的IL2/aOX40-Fc抗体与aPDL1联合使用，发现更好的联合治疗效果依赖于wt Fc(图13B)。(3) In order to further analyze the mechanism of action between aPDL1 and IL2/aOX40-Fc antibody, we used wt Fc or no ADCC Fc IL2/aOX40-Fc antibody in combination with aPDL1, respectively, and found that better combined therapeutic effect depends on wt Fc (FIG. 13B).

实施例5、IL2/aOX40-Fc双功能抗体拮抗TKI治疗后的肿瘤复发Example 5. Tumor recurrence after IL2/aOX40-Fc bifunctional antibody antagonizes TKI therapy

临床上针对EGFR突变肿瘤患者的治疗方案主要是使用靶向受体的抗体或者针对下游信号通路的激酶抑制剂。TKI对于ErBB基因家族相关的肿瘤具有很好的治疗效果，阿法替尼(Afatinib)是一种第二代激酶抑制剂，以共价结合的方式不可逆抑制受体的酪氨酸激酶活性。在TUBO模型上分析了阿法替尼的作用机制，发现治疗后瘤内CD3 T/CD45淋巴细胞的比例上升，说明在化疗药的治疗过程中有更多的T细胞浸润到瘤内(图14A)。单独的Afatinib治疗虽然增加了T细胞向肿瘤的浸润，但不能有效降低瘤内Treg细胞的比例(图14B)。临床上单独的TKI治疗难以完全清除肿瘤，我们推测瘤内的Treg细胞抑制了效应细胞对肿瘤的杀伤，进而导致治疗后的肿瘤复发。The clinical treatment options for patients with EGFR-mutant tumors are mainly the use of antibodies targeting the receptor or kinase inhibitors targeting downstream signaling pathways. TKI has a good therapeutic effect on ErBB gene family-related tumors. Afatinib is a second-generation kinase inhibitor that irreversibly inhibits the tyrosine kinase activity of receptors in a covalent manner. The mechanism of action of afatinib was analyzed on the TUBO model, and it was found that the ratio of CD3 T/CD45 lymphocytes in the tumor increased after treatment, indicating that more T cells infiltrated into the tumor during the treatment of chemotherapy drugs (Fig. 14A). ). Afatinib treatment alone, although increased T cell infiltration into tumors, was not effective in reducing the proportion of intratumoral Treg cells (Figure 14B). Clinically, TKI therapy alone is difficult to completely remove the tumor. We speculate that Treg cells in the tumor inhibit the killing of effector cells on the tumor, which in turn leads to tumor recurrence after treatment.

为了验证IL2/aOX40-Fc是否能够拮抗化疗药治疗后的复发，我们在给荷瘤小鼠化疗药治疗的同时联合使用IL2/aOX40-Fc抗体。In order to verify whether IL2/aOX40-Fc can antagonize the recurrence after chemotherapeutic drug treatment, we used IL2/aOX40-Fc antibody in combination with chemotherapy drug treatment in tumor-bearing mice.

具体实验方案：Specific experimental plan:

BALB/c小鼠皮下接种5×10 ⁵TUBO肿瘤细胞， BALB/c mice were subcutaneously inoculated with 5×10 ⁵ TUBO tumor cells,

治疗方案1：在第9天给予1mg Afatinib灌胃进行治疗，6天后取肿瘤组织进行流式分析。Treatment plan 1: On the 9th day, 1 mg of Afatinib was given by gavage for treatment, and the tumor tissue was taken for flow analysis 6 days later.

治疗方案2：荷瘤小鼠在肿瘤接种后第14天开始治疗，Treatment scheme 2: Tumor-bearing mice started treatment on the 14th day after tumor inoculation,

(1)Afatinib治疗组在第14天灌胃1mg Afatinib，(1) The Afatinib treatment group was given 1 mg of Afatinib on the 14th day,

(2)IL2/aOX40-Fc治疗组则在第14、17和20天腹腔注射25ug抗体，(2) In the IL2/aOX40-Fc treatment group, 25ug of antibody was injected intraperitoneally on the 14th, 17th and 20th days,

(3)联合治疗组按照上述方案Afatinib灌胃一次，25ug IL2/aOX40-Fc腹腔抗体治疗三次。(3) The combined treatment group was administered afatinib once by intragastric administration according to the above scheme, and was treated with 25ug IL2/aOX40-Fc intraperitoneal antibody three times.

结果显示，治疗方案1中的单独的阿法替尼治疗组，肿瘤在得到部分控制后很快复发；而在治疗方案2中，和IL2/aOX40-Fc抗体联合治疗组，可以有效的控制肿瘤的生长，部分小鼠的肿瘤被完全清除(图14C)。The results showed that in the afatinib treatment group alone in treatment plan 1, the tumor relapsed soon after being partially controlled; while in treatment plan 2, the combination treatment group with IL2/aOX40-Fc antibody could effectively control the tumor. tumor growth in some mice was completely cleared (Fig. 14C).

实施例6、IL2/aCTLA4-Fc双特异性抗体显著改善肿瘤治疗效果Example 6. IL2/aCTLA4-Fc bispecific antibody significantly improves tumor treatment effect

体内小鼠荷瘤实验：在C57BL6小鼠皮下接种5×10 ⁵MC38肿瘤细胞，在接种肿瘤后D11开始治疗；分别通过腹腔注射15ug IL2/aCTLA4 wt Fc蛋白(该IL2/aCTLA4 wt Fc蛋白的具体结构示意图如图15所示)。在D11、D14、D17天共治疗三次，每周两次测量肿瘤大小； In vivo mouse tumor-bearing experiment: C57BL6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and treatment was started at D11 after tumor inoculation; 15ug of IL2/aCTLA4 wt Fc protein (the specific A schematic diagram of the structure is shown in Figure 15). Three treatments were performed on D11, D14, and D17, and tumor size was measured twice a week;

结果显示，IL2/aCTLA4-Fc双特异抗体有效控制肿瘤(图15)。The results showed that the IL2/aCTLA4-Fc bispecific antibody effectively controlled tumors (Figure 15).

实施例7、IL2/a4-1BB-Fc双特异性抗体显著改善肿瘤治疗效果Example 7. IL2/a4-1BB-Fc bispecific antibody significantly improves tumor treatment effect

在C57BL/6小鼠皮下接种5×10 ⁵MC38肿瘤细胞，在接种肿瘤后D10开始治疗；分别通过腹腔注射10ug 3H3-IL2和10ug LOB12.3-IL2 wt Fc融合蛋白(IL2/a4-1BB-Fc融合蛋白的结构示意图如图16所示)。在肿瘤接种后的第10、16和19天治疗三次，每周两次测量肿瘤大小；结果显示，与对照组相比，2种融合蛋白的治疗组肿瘤体积明显缩小，差异极显著。 C57BL/6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and treatment was started at D10 after tumor inoculation; 10ug 3H3-IL2 and 10ug LOB12.3-IL2 wt Fc fusion protein (IL2/a4-1BB- A schematic diagram of the structure of the Fc fusion protein is shown in Figure 16). Three treatments were performed on the 10th, 16th and 19th days after tumor inoculation, and the tumor size was measured twice a week; the results showed that compared with the control group, the tumor volume of the treatment group with the two fusion proteins was significantly reduced, and the difference was extremely significant.

实验证明IL2/a4-1BB-Fc融合蛋白具有良好的肿瘤治疗效果(图16)。The experiment proved that the IL2/a4-1BB-Fc fusion protein has a good tumor treatment effect (Fig. 16).

最后需要说明的是，以上实施例仅用作帮助本领域技术人员理解本发明的实质，不用于限制本发明的保护范围。Finally, it should be noted that the above embodiments are only used to help those skilled in the art to understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

[参考文献][references]

[1]MORGAN D A,RUSCETTI F W,GALLO R.Selective in vitro growth of T lymphocytes from normal human bone marrows[J].Science,1976,193(4257):1007-8.[1]MORGAN D A,RUSCETTI F W,GALLO R.Selective in vitro growth of T lymphocytes from normal human bone marrows[J].Science,1976,193(4257):1007-8.

[2]ROBB R J,SMITH K A.Heterogeneity of human T-cell growth factor(s)due to variable glycosylation[J].Molecular immunology,1981,18(12):1087-94.[2] ROBB R J, SMITH K A.Heterogeneity of human T-cell growth factor(s) due to variable glycosylation[J].Molecular immunology,1981,18(12):1087-94.

[3]TANIGUCHI T,MINAMI Y.The IL-2IL-2 receptor system:a current overview[J].Cell,1993,73(1):5-8.[3]TANIGUCHI T,MINAMI Y.The IL-2IL-2 receptor system:a current overview[J].Cell,1993,73(1):5-8.

[4]MALEK T R.The biology of interleukin-2[J].Annu Rev Immunol,2008,26(453-79.[4]MALEK T R.The biology of interleukin-2[J].Annu Rev Immunol,2008,26(453-79.

[5]LEONARD W J,DEPPER J M,CRABTREE G R,et al.Molecular cloning and expression of cDNAs for the human interleukin-2 receptor[J].Nature,1984,311(5987):626-31.[5]LEONARD W J, DEPPER J M, CRABTREE G R, et al.Molecular cloning and expression of cDNAs for the human interleukin-2 receptor[J].Nature,1984,311(5987):626-31.

[6]NIKAIDO T,SHIMIZU A,ISHIDA N,et al.Molecular cloning of cDNA encoding human interleukin-2 receptor[J].Nature,1984,311(5987):631-5.[6] NIKAIDO T, SHIMIZU A, ISHIDA N, et al.Molecular cloning of cDNA encoding human interleukin-2 receptor[J].Nature,1984,311(5987):631-5.

[7]LIAO W,LIN J-X,LEONARD W J.Interleukin-2 at the crossroads of effector responses,tolerance,and immunotherapy[J].Immunity,2013,38(1):13-25.[7]LIAO W,LIN J-X,LEONARD W J.Interleukin-2 at the crossroads of effector responses,tolerance,and immunotherapy[J].Immunity,2013,38(1):13-25.

[8]FLYNN M J,HARTLEY J A.The emerging role of anti‐CD 25 directed therapies as both immune modulators and targeted agents in cancer[J].British journal of haematology,2017,179(1):20-35.[8] FLYNN M J, HARTLEY J A. The emerging role of anti‐CD 25 directed therapies as both immune modulators and targeted agents in cancer[J].British journal of haematology,2017,179(1):20-35.

[9]FONTENOT J D,RASMUSSEN J P,GAVIN M A,et al.A function for interleukin 2 in Foxp3-expressing regulatory T cells[J].Nature immunology,2005,6(11):1142-51.[9] FONTENOT J D, RASMUSSEN J P, GAVIN M A, et al.A function for interleukin 2 in Foxp3-expressing regulatory T cells[J].Nature immunology,2005,6(11):1142-51.

[10]SIEGEL J P,SHARON M,SMITH P L,et al.The IL-2 receptor beta chain(p70):role in mediating signals for LAK,NK,and proliferative activities[J].Science,1987,238(4823):75-8.[10] SIEGEL J P, SHARON M, SMITH P L, et al. The IL-2 receptor beta chain (p70): role in mediating signals for LAK, NK, and proliferative activities[J].Science,1987,238( 4823):75-8.

[11]CHARYCH D,KHALILI S,DIXIT V,et al.Modeling the receptor pharmacology,pharmacokinetics,and pharmacodynamics of NKTR-214,a kinetically-controlled interleukin-2(IL2)receptor agonist for cancer immunotherapy[J].PloS one,2017,12(7):e0179431.[11]CHARYCH D, KHALILI S, DIXIT V, et al. Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy [J]. PloS one ,2017,12(7):e0179431.

[12]LEVIN A M,BATES D L,RING A M,et al.Exploiting a natural conformational switch to engineer an interleukin-2‘superkine’[J].Nature,2012,484(7395):529-33.[12] LEVIN A M, BATES D L, RING A M, et al.Exploiting a natural conformational switch to engineer an interleukin-2‘superkine’[J].Nature,2012,484(7395):529-33.

[13]SUN Z,REN Z,YANG K,et al.A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+T-cell response and effective tumor control[J].Nature communications,2019,10(1):1-12.[13]SUN Z,REN Z,YANG K,et al.A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+T-cell response and effective tumor control[J].Nature communications,2019,10 (1): 1-12.

[14]MARABELLE A,KOHRT H,SAGIV-BARFI I,et al.Depleting tumor-specific Tregs at a single site eradicates disseminated tumors[J].The Journal of clinical investigation,2013,123(6):2447-63.[14] MARABELLE A, KOHRT H, SAGIV-BARFI I, et al.Depleting tumor-specific Tregs at a single site eradicates disseminated tumors[J].The Journal of clinical investigation,2013,123(6):2447-63.

[15]CURTI B D,KOVACSOVICS-BANKOWSKI M,MORRIS N,et al.OX40 is a potent immune-stimulating target in late-stage cancer patients[J].Cancer research,2013,73(24):7189-98.[15] CURTI B D, KOVACSOVICS-BANKOWSKI M, MORRIS N, et al. OX40 is a potent immune-stimulating target in late-stage cancer patients[J]. Cancer research, 2013, 73(24): 7189-98.

[16]SONG A,SONG J,TANG X,et al.Cooperation between CD4 and CD8 T cells for anti‐tumor activity is enhanced by OX40 signals[J].European journal of immunology,2007,37(5):1224-32.[16]SONG A,SONG J,TANG X,et al.Cooperation between CD4 and CD8 T cells for anti‐tumor activity is enhanced by OX40 signals[J].European journal of immunology,2007,37(5):1224- 32.

[17]BULLIARD Y,JOLICOEUR R,ZHANG J,et al.OX40 engagement depletes intratumoral Tregs via activating FcγRs,leading to antitumor efficacy[J].Immunology and cell biology,2014,92(6):475-80.[17] BULLIARD Y, JOLICOEUR R, ZHANG J, et al. OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy [J]. Immunology and cell biology, 2014, 92(6): 475-80.

[18]LINCH S N,MCNAMARA M J,REDMOND W L.OX40 agonists and combination immunotherapy:putting the pedal to the metal[J].Frontiers in oncology,2015,5(34).[18]LINCH S N, MCNAMARA M J, REDMOND W L. OX40 agonists and combination immunotherapy: putting the pedal to the metal [J]. Frontiers in oncology, 2015, 5(34).

[19]CHIBA K.FTY720,a new class of immunomodulator,inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors[J].Pharmacology&therapeutics,2005,108(3):308-19.[19] CHIBA K.FTY720, a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors[J].Pharmacology&therapeutics,2005,108(3):308-19.

Claims

一种双功能融合蛋白，所述的融合蛋白为异源二聚体，其特征在于，A bifunctional fusion protein, the fusion protein is a heterodimer, characterized in that,

所述的异源二聚体包括：Described heterodimer includes:

(1)白介素2(IL-2)与免疫球蛋白Fc单链连接而成的第一单体；(1) The first monomer formed by linking interleukin 2 (IL-2) and immunoglobulin Fc single chain;

(2)抗CTLA4或TNF家族的共刺激或共抑制性分子的抗体的Fab或ScFv与免疫球蛋白Fc单链连接而成的第二单体；(2) a second monomer formed by linking the Fab or ScFv of an antibody against a co-stimulatory or co-inhibitory molecule of the CTLA4 or TNF family to an immunoglobulin Fc single chain;

所述的第一单体与第二单体通过Fc单链的二聚化连接，构成所述的异源二聚体；The first monomer and the second monomer are connected by dimerization of the Fc single chain to form the heterodimer;

所述的CTLA4或TNF家族的共刺激或共抑制性分子包括但不限于：OX40、ICOS、GITR、4-1BB。The co-stimulatory or co-inhibitory molecules of CTLA4 or TNF family include but are not limited to: OX40, ICOS, GITR, 4-1BB.
根据权利要求1所述的双功能融合蛋白，其特征在于，所述的免疫球蛋白Fc单链为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链；优选的，所述的免疫球蛋白Fc单链为人IgG的Fc单链。The bifunctional fusion protein according to claim 1, wherein the immunoglobulin Fc single chain is a natural immunoglobulin Fc single chain or an immunoglobulin Fc single chain with ADCC effect knocked out by gene mutation; preferably The said immunoglobulin Fc single chain is the Fc single chain of human IgG.
根据权利要求1任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claim 1, wherein,

所述的第二单体中，In the second monomer,

所述的抗体的Fab为人源化抗体的Fab或全人化抗体的Fab；The Fab of the antibody is the Fab of a humanized antibody or the Fab of a fully humanized antibody;

所述的抗体的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；The ScFv of the antibody is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;

优选的，所述的第二单体为：Preferably, the second monomer is:

抗CTLA4或TNF家族的共刺激或共抑制性分子的抗体的单体，所述抗体的单体包含一条轻链和一条重链；A monomer of an antibody against a co-stimulatory or co-inhibitory molecule of the CTLA4 or TNF family, the monomer of which comprises one light chain and one heavy chain;

更优选的，所述的第二单体为：More preferably, the second monomer is:

人源化抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体的单体或全人化抗CTLA4或抗TNF家族的共刺激或共抑制性分子的抗体抗体的单体，所述抗体的单体包含一条轻链和一条重链。Monomer of a humanized anti-CTLA4 or antibody against a costimulatory or costinhibitory molecule of the TNF family or a monomer of a fully humanized antibody against a costimulatory or costinhibitory molecule of the TNF family, said antibody The monomer contains one light chain and one heavy chain.
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的CTLA4或TNF家族的共刺激或共抑制性分子为OX40、4-1BB，The co-stimulatory or co-inhibitory molecules of the CTLA4 or TNF family are OX40, 4-1BB,

所述的抗CTLA4的抗体(aCTLA4)或抗TNF家族的共刺激或共抑制性分子的抗体为抗OX40的抗体(aOX40)、或抗4-1BB的抗体(a4-1BB)。The anti-CTLA4 antibody (aCTLA4) or the anti-TNF family co-stimulatory or co-inhibitory molecule antibody is an anti-OX40 antibody (aOX40) or an anti-4-1BB antibody (a4-1BB).
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的异源二聚体包括：Described heterodimer includes:

(1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus:

1)序列如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) The wild-type IL-2 protein whose sequence is shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, the mutant comprising any one or any combination of the following mutation sites ;F42A, L80F, R81D, L85V, I86V and I92F;

2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) necessary connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;

3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) The Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the knob mutation shown in SEQ ID NO.4 type Fc, or hole mutant Fc as shown in SEQ ID NO.5;

(2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises:

1)序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL和序列如SEQ ID NO.8所示的抗OX40抗体重链VH&CH1组成的抗OX40抗体Fab区；1) the anti-OX40 antibody Fab region composed of the anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7 and the anti-OX40 antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.8;

或：2)序列如SEQ ID NO.9所示的抗OX40单链抗体(ScFv)：Or: 2) anti-OX40 single chain antibody (ScFv) whose sequence is shown in SEQ ID NO.9:

和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 A knob mutant Fc, or a hole mutant Fc as shown in SEQ ID NO.5.
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的异源二聚体包括：Described heterodimer includes:

第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;

第二单体：其为：Second monomer: which is:

(1)序列如SEQ ID NO.11所示的抗OX40抗体VH-CH1-Fc(knob)和序列如SEQ ID NO.7所示的抗OX40抗体轻链VL-KCL组成；(1) the anti-OX40 antibody VH-CH1-Fc (knob) whose sequence is shown in SEQ ID NO.11 and the anti-OX40 antibody light chain VL-KCL whose sequence is shown in SEQ ID NO.7;

或(2)序列如SEQ ID NO.12所示的多肽(aOX40 ScFv-Fc(knob))。。Or (2) a polypeptide whose sequence is shown in SEQ ID NO. 12 (aOX40 ScFv-Fc(knob)). .
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的异源二聚体包括：Described heterodimer includes:

(1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus:

1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) a wild-type IL-2 protein as shown in SEQ ID NO.1, or a mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites; F42A, L80F, R81D, L85V, I86V and I92F;

2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) necessary connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;

3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) Fc single chain of IgG as shown in SEQ ID NO.2, or Fc of No-ADCC mutant IgG as shown in SEQ ID NO.3, or knob mutant as shown in SEQ ID NO.4 Fc, or a hole mutant Fc as shown in SEQ ID NO.5;

(2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises:

1)序列如SEQ ID NO.21所示的抗CTLA4抗体轻链与序列如SEQ ID NO.22所示的抗CTLA4抗体重链VH&CH1组成的抗体Fab区；1) the antibody Fab region composed of the anti-CTLA4 antibody light chain whose sequence is shown in SEQ ID NO.21 and the anti-CTLA4 antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.22;

或：2)序列如SEQ ID NO.23所示的抗CTLA4单链抗体(ScFv)：Or: 2) anti-CTLA4 single-chain antibody (ScFv) whose sequence is shown in SEQ ID NO.23:

和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 A knob mutant Fc, or a hole mutant Fc as shown in SEQ ID NO.5.
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的异源二聚体包括：Described heterodimer includes:

第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;

第二单体：其为：Second monomer: which is:

(1)序列如SEQ ID NO.24所示的多肽(aCTLA4 VH-CH1-Fc(knob))和序列如SEQ ID NO.21所示的抗CTLA4抗体轻链可变区组成；或(1) The polypeptide (aCTLA4 VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.24 and the light chain variable region of the anti-CTLA4 antibody whose sequence is shown in SEQ ID NO.21; or

(2)序列如SEQ ID NO.25所示的多肽(a CTLA4 ScFv-Fc(knob))。(2) The polypeptide (a CTLA4 ScFv-Fc(knob)) whose sequence is shown in SEQ ID NO.25.
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的异源二聚体包括：Described heterodimer includes:

(1)第一单体，所述的第一单体自N端开始依次包含：(1) the first monomer, the first monomer comprises sequentially from the N-terminus:

1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；1) a wild-type IL-2 protein as shown in SEQ ID NO.1, or a mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites; F42A, L80F, R81D, L85V, I86V and I92F;

2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.6所示；2) necessary connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.6;

3)如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc；3) Fc single chain of IgG as shown in SEQ ID NO.2, or Fc of No-ADCC mutant IgG as shown in SEQ ID NO.3, or knob mutant as shown in SEQ ID NO.4 Fc, or a hole mutant Fc as shown in SEQ ID NO.5;

(2)第二单体，所述的第二单体包含：(2) a second monomer, the second monomer comprises:

1)序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链与序列如SEQ ID NO.30、31所示的抗4-1BB 抗体重链VH&CH1组成的抗体Fab区；1) the antibody Fab region composed of the anti-4-1BB antibody light chain whose sequence is shown in SEQ ID NO.28 and 29 and the anti-4-1BB antibody heavy chain VH&CH1 whose sequence is shown in SEQ ID NO.30 and 31;

或：2)序列如SEQ ID NO.32、33所示的抗4-1BB单链抗体(ScFv)：Or: 2) anti-4-1BB single chain antibody (ScFv) whose sequence is shown in SEQ ID NO.32, 33:

和：3)序列如SEQ ID NO.2所示的IgG的Fc单链、或如SEQ ID NO.3所示的No-ADCC突变型的IgG的Fc、或如SEQ ID NO.4所示的knob突变型Fc、或如SEQ ID NO.5所示的hole突变型Fc。and: 3) the Fc single chain of IgG whose sequence is shown in SEQ ID NO.2, or the Fc of No-ADCC mutant IgG shown in SEQ ID NO.3, or the Fc shown in SEQ ID NO.4 A knob mutant Fc, or a hole mutant Fc as shown in SEQ ID NO.5.
根据权利要求1-3任一所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to any one of claims 1-3, wherein,

所述的异源二聚体包括：Described heterodimer includes:

第一单体：其为序列如SEQ ID NO.10所示的多肽(IL2-Fc)；The first monomer: it is a polypeptide (IL2-Fc) whose sequence is shown in SEQ ID NO.10;

第二单体：其为：Second monomer: which is:

(1)序列如SEQ ID NO.34、35所示的多肽(a4-1BB VH-CH1-Fc(knob))和序列如SEQ ID NO.28、29所示的抗4-1BB抗体轻链组成；或(1) The composition of the polypeptide (a4-1BB VH-CH1-Fc(knob)) whose sequence is shown in SEQ ID NO.34 and 35 and the light chain of anti-4-1BB antibody whose sequence is shown in SEQ ID NO.28 and 29 ;or

(2)序列如SEQ ID NO.36、37所示的多肽(a4-1BB ScFv-Fc(knob))。(2) The polypeptide (a4-1BB ScFv-Fc(knob)) whose sequence is shown in SEQ ID NO.36 and 37.
一种双功能融合蛋白，所述的融合蛋白为同源二聚体，其特征在于，A bifunctional fusion protein, the fusion protein is a homodimer, characterized in that,

所述的同源二聚体的单体为：The monomer of the homodimer is:

一分子白介素2(IL-2)与一分子抗OX40的抗体(aOX40)、抗CTLA4的抗体(aCTLA4)或抗4-1BB的抗体(a4-1BB)的Fab通过任意方式连接构成的单体，或，A molecule of interleukin 2 (IL-2) and a molecule of anti-OX40 antibody (aOX40), anti-CTLA4 antibody (aCTLA4) or anti-4-1BB antibody (a4-1BB) Fab linked in any way to form a monomer, or,

一分子白介素2(IL-2)与一分子抗OX40的抗体(aOX40)、抗CTLA4的抗体(aCTLA4)或抗4-1BB的抗体(a4-1BB)的(ScFv)通过任意方式连接构成的单体。A single molecule of interleukin 2 (IL-2) linked to a molecule of anti-OX40 antibody (aOX40), anti-CTLA4 antibody (aCTLA4) or anti-4-1BB antibody (a4-1BB) (ScFv) by any means body.
根据权利要求11所述的双功能融合蛋白，其特征在于，The bifunctional fusion protein according to claim 11, wherein,

所述的同源二聚体的单体自N端开始依次包含：The monomers of the homodimer, starting from the N-terminus, sequentially include:

(1)如SEQ ID NO.1所示的野生型IL-2蛋白、或所述的野生型IL-2蛋白的突变体，所述的突变体中包含如下任一或任意组合的突变位点；F42A、L80F、R81D、L85V、I86V和I92F；(1) The wild-type IL-2 protein shown in SEQ ID NO.1, or the mutant of the wild-type IL-2 protein, wherein the mutant comprises any one or any combination of the following mutation sites ;F42A, L80F, R81D, L85V, I86V and I92F;

(2)必要的连接结构(G4S连接序列)，优选的，所述的连接序列如SEQ ID NO.4、SEQ ID NO.5所示；(2) necessary connection structure (G4S connection sequence), preferably, described connection sequence is as shown in SEQ ID NO.4, SEQ ID NO.5;

(3)抗OX40的抗体(aOX40)的Fab/ScFv、抗CTLA4的抗体(aCTLA4)的Fab/ScFv或抗4-1BB的抗体(a4-1BB)的Fab/ScFv；所述的Fab为人源化抗体的Fab或全人化抗体的Fab，所述的ScFv为人源化抗体的ScFv或全人化抗体的ScFv；(3) Fab/ScFv of anti-OX40 antibody (aOX40), Fab/ScFv of anti-CTLA4 antibody (aCTLA4) or Fab/ScFv of anti-4-1BB antibody (a4-1BB); the Fab is humanized The Fab of an antibody or the Fab of a fully humanized antibody, the ScFv is the ScFv of a humanized antibody or the ScFv of a fully humanized antibody;

(4)抗体的Fc；所述的抗体Fc为全人野生型Fc或No-ADCC突变型Fc。(4) Fc of the antibody; the Fc of the antibody is a fully human wild-type Fc or a No-ADCC mutant Fc.
编码所述权利要求1-12任一所述的双功能融合蛋白的核苷酸序列。The nucleotide sequence encoding the bifunctional fusion protein of any one of claims 1-12.
根据权利要求13所述的核苷酸序列，其特征在于，The nucleotide sequence of claim 13, wherein

编码所述异源二聚体第一单体的核苷酸序列为SEQ ID NO.11所示的核苷酸序列；The nucleotide sequence encoding the first monomer of the heterodimer is the nucleotide sequence shown in SEQ ID NO.11;

编码所述异源二聚体第二单体的核苷酸序列为：The nucleotide sequence encoding the second monomer of the heterodimer is:

SEQ ID NO.16(aOX40：VL-KCL)、SEQ ID NO. 16 (aOX40:VL-KCL),

SEQ ID NO.17(aOX40：VH-CH1-Fc(knob))、SEQ ID NO. 17 (aOX40: VH-CH1-Fc(knob)),

SEQ ID NO.18(aOX40 ScFv-Fc(knob))、SEQ ID NO. 18 (aOX40 ScFv-Fc(knob)),

SEQ ID NO.42(a4-1BB(LOB12.3)：VL-KCL)、SEQ ID NO.42 (a4-1BB(LOB12.3):VL-KCL),

SEQ ID NO.43(a4-1BB(3H3)：VL-KCL)、SEQ ID NO.43 (a4-1BB(3H3):VL-KCL),

SEQ ID NO.44(a4-1BB(LOB12.3)：VH-CH1-Fc(knob))、SEQ ID NO. 44 (a4-1BB(LOB12.3): VH-CH1-Fc(knob)),

SEQ ID NO.45(a4-1BB(3H3)：VH-CH1-Fc(knob))、SEQ ID NO. 45 (a4-1BB(3H3): VH-CH1-Fc(knob)),

SEQ ID NO.46(a4-1BB(LOB12.3)：ScFv-Fc(knob))、SEQ ID NO. 46 (a4-1BB(LOB12.3): ScFv-Fc(knob)),

SEQ ID NO.47(a4-1BB(3H3)：ScFv-Fc(knob))所示的核苷酸序列；The nucleotide sequence shown in SEQ ID NO.47 (a4-1BB(3H3): ScFv-Fc(knob));

编码所述同源二聚体的核苷酸序列为：The nucleotide sequence encoding the homodimer is:

SEQ ID NO.19(aOX40-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 19 (aOX40-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.20(IL2-aOX40：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 20 (IL2-aOX40: IL2-VL-VH(ScFv)-Fc(wt)),

SEQ ID NO.48(a4-1BB(LOB12.3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 48 (a4-1BB(LOB12.3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.49(a4-1BB(3H3)-IL2：VL-VH(ScFv)-Fc(wt)-IL2)、SEQ ID NO. 49 (a4-1BB(3H3)-IL2:VL-VH(ScFv)-Fc(wt)-IL2),

SEQ ID NO.50(IL2-a4-1BB(LOB12.3)：IL2-VL-VH(ScFv)-Fc(wt))、SEQ ID NO. 50 (IL2-a4-1BB(LOB12.3):IL2-VL-VH(ScFv)-Fc(wt)),

SEQ ID NO.51(IL2-a4-1BB(3H3)：IL2-VL-VH(ScFv)-Fc(wt))所示的核苷酸序列。The nucleotide sequence shown in SEQ ID NO. 51 (IL2-a4-1BB(3H3):IL2-VL-VH(ScFv)-Fc(wt)).
权利要求1-12任一所述的双功能融合蛋白的如下应用：The following application of the bifunctional fusion protein of any one of claims 1-12:

(1)制备抗肿瘤药物；(1) Preparation of antitumor drugs;

(2)制备与免疫检查点抑制剂联用的抗肿瘤药物；(2) Preparation of anti-tumor drugs combined with immune checkpoint inhibitors;

(3)制备克服免疫检查点抑制剂耐受的抗肿瘤药物；(3) Preparation of anti-tumor drugs that overcome immune checkpoint inhibitor tolerance;

(4)制备与TKI拮抗剂联用的抗肿瘤药物；(4) preparation of antitumor drugs combined with TKI antagonists;

(5)制备克服TKI拮抗剂耐受的抗肿瘤药物。(5) Preparation of antitumor drugs that overcome TKI antagonist tolerance.
根据权利要求15所述的应用，其特征在于，所述的免疫检查点抑制剂为抗PD-L1的拮抗剂，优选的，所述的抗PD-L1的拮抗剂为抗PD-L1的抗体。The application according to claim 15, wherein the immune checkpoint inhibitor is an anti-PD-L1 antagonist, preferably, the anti-PD-L1 antagonist is an anti-PD-L1 antibody .
根据权利要求15所述的应用，其特征在于，所述的TKI拮抗剂为小分子TKI拮抗剂；优选的，所述的小分子TKI拮抗剂为阿法替尼或其结构类似物。The application according to claim 15, wherein the TKI antagonist is a small molecule TKI antagonist; preferably, the small molecule TKI antagonist is afatinib or a structural analog thereof.
包含权利要求1-12任一所述的双功能融合蛋白的药物或药物组合物。A medicine or a pharmaceutical composition comprising the bifunctional fusion protein of any one of claims 1-12.